Sequences and their use for detection and characterization of e. coli 0157:h7

ABSTRACT

This invention relates to a rapid method for detection and characterization of  Escherichia coli  bacteria serotype O157:H7 based on the presence of nucleic acid sequences, in particular, to a PCR-based method for detection, and to oligonucleotide molecules and reagents and kits useful therefore. This method is preferably employed to detect  E. coli  O157:H7 in a food or water sample, such as a beef enrichment. The present invention further relates to replication compositions and kits for carrying out the method of the present invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/227,622, filed Jul. 22, 2009, which is incorporated by referenceherein in its entirety.

GOVERNMENT INTERESTS

This invention was made under a Cooperative Research and DevelopmentAgreement with the Agricultural Research Service (Agreement No.58-3K95-8-1254-M). The government may have certain rights in thisinvention.

FIELD OF INVENTION

The field of invention relates to methods for detection andcharacterization of Escherichia coli bacteria serotype O157:H7 based onthe presence of nucleic acid sequences, preferably PCR-based methods fordetection, and to oligonucleotide molecules and reagents and kits usefultherefor.

BACKGROUND OF INVENTION

Escherichia coli (E. coli) is a gram-negative, rod-shaped bacterium.Although most strains of E. coli are benign and are found as normalintestinal flora of humans and other animals, some strains arepathogenic and can lead to sometimes-fatal disease. Different strains ofpathogenic E. coli differ in their epidemiology, clinical course andpotential for causing outbreaks of disease. Passage of disease isgenerally through the fecal/oral route.

Pathogenicity has been linked to several serotypes, as defined by O andH antigens. Different pathogenic serotypes are associated with differentclinical disease courses and have associated with them different levelsof concern from the standpoint of public health. Several outbreaks ofdisease have been tracked to food and water borne sources of pathogenicE. coli.

One serotype of E. coli in particular, serotype O157:H7, has beenassociated with several food and water borne outbreaks and is regulatedas an adulterant in ground beef by the U.S. Department of Agriculture(USDA) with a zero tolerance standard. This serotype of E. coli isbelieved to have arisen from an O55:H7 parent strain, which thenswitched from 055 to O157 upon the transfer into the progenitor O55:H7genome of the large virulence plasmid pO157, which contained theO157-rfb gene cluster as well as some additional genetic information(see, e.g., Lukas M. Wick, et al., Evolution of Genomic Content in theStepwise Emergence of Escherichia coli O157:H7, Journal of Bacteriology187:1783-91 (2005)).

Since E. coli is ubiquitous, and since serotype O157:H7 is highlypathogenic and tightly regulated, the ability to specifically detect andcharacterize E. coli serotype O157:H7 in a sample, even in the presenceof other E. coli serotypes, is useful.

It is desirable, therefore, to have a test for the accurate detectionand characterization of E. coli O157:H7 in a sample.

SUMMARY OF INVENTION

One aspect is for a method for detecting the presence of E. coli O157:H7in a sample, said sample comprising nucleic acids, said methodcomprising:

-   -   (a) providing a reaction mixture comprising suitable primer        pairs for amplification of at least a portion of        -   (i) one or more E. coli O157:H7 genomic DNA regions within            the pO157 portion of the E. coli O157:H7 genome, and        -   (ii) one or more E. coli O157:H7 genomic DNA regions outside            the pO157 portion of the E. coli O157:H7 genome;    -   (b) performing PCR amplification of said nucleic acids of said        sample using the reaction mixture of step (a); and    -   (c) detecting the amplification of step (b), whereby a positive        detection of amplification indicates the presence of E. coli        O157:H7 in the sample.

Another aspect is for an isolated polynucleotide comprising SEQ ID NO:4,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, or SEQ IDNO:26.

A further aspect is for a replication composition for use in performanceof PCR, comprising:

-   -   (a) a primer pair comprising nucleic acid sequences SEQ ID NO:4        and SEQ ID NO:5;    -   (b) one or more primer pairs comprising nucleic acid sequences        selected from the group consisting of:        -   (i) SEQ ID NO:10 and SEQ ID NO:12;        -   (ii) SEQ ID NO:15 and SEQ ID NO:16; and        -   (iii) a combination thereof; and    -   (b) thermostable DNA polymerase.

An additional aspect is for a replication composition for use inperformance of PCR, comprising:

-   -   (a) a primer pair comprising nucleic acid sequences SEQ ID NO:5        and SEQ ID NO:6;    -   (b) one or more primer pairs comprising nucleic acid sequences        selected from the group consisting of:        -   (i) SEQ ID NO:12 and SEQ ID NO:13;        -   (ii) SEQ ID NO:16 and SEQ ID NO:17; and        -   (iii) a combination thereof; and    -   (b) thermostable DNA polymerase.

Other objects and advantages will become apparent to those skilled inthe art upon reference to the detailed description that hereinafterfollows.

SUMMARY OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of a region outside the pO157portion of E. coli serotype O157:H7 genome, the detection of whichspecifically shows the presence of that bacterial serotype. SEQ ID NO:1or its reverse complementary sequence is bounded by, contains orcontains binding sites for, and can be amplified by the use of SEQ IDNO:5 or SEQ ID NO:8 in conjunction with SEQ ID NO:4 or SEQ ID NO:6. Thisregion also contains a binding site for the oligonucleotide SEQ ID NO:7or SEQ ID NO:9.

SEQ ID NO:2 is the nucleotide sequence of a region within the pO157portion of E. coli serotype O157:H7 genome, the detection of whichspecifically shows the presence of that bacterial serotype. SEQ ID NO:2or its reverse complementary sequence is bounded by, contains orcontains binding sites for, and can be amplified by the use of SEQ IDNO:12 in conjunction with SEQ ID NO:10 or SEQ ID NO:13, and alsocontains a binding site for probe SEQ ID NO:14.

SEQ ID NO:3 is the nucleotide sequence of a region within the pO157portion of E. coli serotype O157:H7 genome, the detection of whichspecifically shows the presence of that bacterial serotype. SEQ ID NO:3or its reverse complementary sequence is bounded by, contains orcontains binding sites for, and can be amplified by the use of SEQ IDNO:16 in conjunction with SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19,and also contains a binding site for SEQ ID NO:18 and SEQ ID NO:20.

SEQ ID NO:4 is the nucleotide sequence of a primer-probe complex fordetection of E. coli serotype O157:H7, specifically the region of thegenome identified by SEQ ID NO:1. The 3′ portion of this primer-probecomplex is capable of hybridizing to SEQ ID NO:1 or its reversecomplementary sequence and acting as a 5′ primer, such as during anamplification reaction that also employs an appropriate 3′ primer, suchas SEQ ID NO:5. The 5′ portion of this primer-probe complex contains asegment capable of hybridizing to SEQ ID NO:1 or its reversecomplementary sequence at a location downstream (i.e., in the 3′direction) from the binding location of the primer portion of theprimer-probe complex, and upstream (i.e., in the 5′ direction) of thebinding location of any 3′ primer that is employed in an amplificationreaction. The 5′ portion of this primer-probe complex also contains twoself-complementary segments (nucleotides 1-9 and 38-46) capable ofself-hybridizing to form a stem-loop structure. The 5′ and 3′ portionsof this primer-probe complex are separated by a non-amplifiable linkerbetween nucleotide 46 (“T”) and nucleotide 47 (“G”). Thisnon-amplifiable linker is capable of blocking elongation of acomplementary strand to this primer-probe complex.

SEQ ID NO:5 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:1 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 3′ primer with anappropriate 5′ primer, such as SEQ ID NO:4 or SEQ ID NO:6.

SEQ ID NO:6 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:1 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 5′ primer with anappropriate 3′ primer, such as SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:1 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a probe thatwill hybridize to and allow detection of the DNA of that bacterialserotype in a polymerase chain reaction with bacterial DNA when usedwith an appropriate 5′ primer, such as SEQ ID NO:6, and an appropriate3′ primer, such as SEQ ID NO:5.

SEQ ID NO:8 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:1 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 3′ primer with anappropriate 5′ primer, such as SEQ ID NO:4 or SEQ ID NO:6.

SEQ ID NO:9 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:1 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 5′ primer with anappropriate 3′ primer.

SEQ ID NO:10 is the nucleotide sequence of a primer-probe complex fordetection of E. coli serotype O157:H7, specifically the region of thegenome identified by SEQ ID NO:2. The 3′ portion of this primer-probecomplex is capable of hybridizing to SEQ ID NO:2 or its reversecomplement sequence and acting as a 5′ primer, such as during anamplification reaction that also employs an appropriate 3′ primer, suchas SEQ ID NO:12. The 5′ portion of this primer-probe complex contains asegment capable of hybridizing to SEQ ID NO:2 or its reverse complementsequence at a location downstream (i.e., in the 3′ direction) from thebinding location of the primer portion of the primer-probe complex, andpreferably upstream (i.e., in the 5′ direction) of the binding locationof any 3′ primer that is employed in an amplification reaction. The 5′and 3′ portions of this primer-probe complex are separated by anon-amplifiable linker between nucleotide 34 (“G”) and nucleotide 35(“C”). This non-amplifiable linker is capable of blocking elongation ofa complementary strand to this primer-probe complex.

SEQ ID NO:11 is a blocking oligonucleotide capable of hybridizing to theprobe portion of the SEQ ID NO:10 probe-primer complex.

SEQ ID NO:12 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:2 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 3′ primer with anappropriate 5′ primer, such as SEQ ID NO:10 or SEQ ID NO:13.

SEQ ID NO:13 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:2 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 5′ primer with anappropriate 3′ primer, such as SEQ ID NO:12.

SEQ ID NO:14 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:2 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a probe thatwill hybridize to and allow detection of the DNA of that bacterialserotype in a polymerase chain reaction with bacterial DNA when usedwith an appropriate 5′ primer, such as SEQ ID NO:13, and an appropriate3′ primer, such as SEQ ID NO:12.

SEQ ID NO:15 is the nucleotide sequence of a primer-probe complex fordetection of E. coli serotype O157:H7, specifically the region of thegenome identified by SEQ ID NO:3. The 3′ portion of this primer-probecomplex is capable of hybridizing to SEQ ID NO:3 or its reversecomplement sequence and acting as a 5′ primer, such as during anamplification reaction that also employs an appropriate 3′ primer, suchas SEQ ID NO:16. The 5′ portion of this primer-probe complex contains asegment capable of hybridizing to SEQ ID NO:3 or its reverse complementsequence at a location downstream (i.e., in the 3′ direction) from thebinding location of the primer portion of the primer-probe complex, andpreferably upstream (i.e., in the 5′ direction) of the binding locationof any 3′ primer that is employed in an amplification reaction. The 5′portion of this primer-probe complex also contains twoself-complementary segments (nucleotides 1-9 and 37-45) capable ofself-hybridizing to form a stem-loop structure. The 5′ and 3′ portionsof this primer-probe complex are separated by a non-amplifiable linkerbetween nucleotide 45 (“T”) and nucleotide 46 (“A”). Thisnon-amplifiable linker is capable of blocking elongation of acomplementary strand to this primer-probe complex.

SEQ ID NO:16 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:3 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 3′ primer with anappropriate 5′ primer, such as SEQ ID NO:15 or SEQ ID NO:17.

SEQ ID NO:17 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:3 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 5′ primer with anappropriate 3′ primer, such as SEQ ID NO:16.

SEQ ID NO:18 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:3 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a probe thatwill hybridize to and allow detection of the DNA of that bacterialserotype in a polymerase chain reaction with bacterial DNA when usedwith an appropriate 5′ primer, such as SEQ ID NO:17, and an appropriate3′ primer, such as SEQ ID NO:16.

SEQ ID NO:19 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:3 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 5′ primer with anappropriate 3′ primer, such as SEQ ID NO: 16.

SEQ ID NO:20 is the nucleotide sequence of a region of the genome of E.coli serotype O157:H7 capable for use as a probe or primer in detectionof the presence of SEQ ID NO:3 and subsequent detection of the presenceof E. coli O157:H7. This sequence is preferably used as a primer thatwill specifically amplify DNA of that bacterial serotype in a polymerasechain reaction with bacterial DNA when used as a 3′ primer with anappropriate 5′ primer.

SEQ ID NO:21 is the nucleotide sequence capable for use as an internalcontrol primer. This sequence is preferably used as a primer that willspecifically amplify DNA in a polymerase chain reaction with a controltemplate DNA when used as a 5′ primer with an appropriate 3′ primer,such as SEQ ID NO:22.

SEQ ID NO:22 is the nucleotide sequence capable for use as an internalcontrol primer. This sequence is preferably used as a primer that willspecifically amplify DNA in a polymerase chain reaction with a controltemplate DNA when used as a 3′ primer with an appropriate 5′ primer,such as SEQ ID NO:21.

SEQ ID NO:23 is the nucleotide sequence of an internal control Taqman®probe.

SEQ ID NO:24 is the nucleotide sequence of a Taqman® probe for detectionof E. coli serotype O157:H7, specifically the region of the genomeidentified by SEQ ID NO:2.

SEQ ID NO:25 is the nucleotide sequence of a Taqman® probe for detectionof E. coli serotype O157:H7, specifically the region of the genomeidentified by SEQ ID NO:1.

SEQ ID NO:26 is the nucleotide sequence of a Taqman® probe for detectionof E. coli serotype O157:H7, specifically the region of the genomeidentified by SEQ ID NO:3.

SEQ ID NO:27 is the nucleotide sequence capable for use as an internalcontrol primer. This sequence is preferably used as a primer that willspecifically amplify DNA in a polymerase chain reaction with a controltemplate DNA when used as a 5′ primer with an appropriate 3′ primer,such as SEQ ID NO:28.

SEQ ID NO:28 is the nucleotide sequence capable for use as an internalcontrol primer. This sequence is preferably used as a primer that willspecifically amplify DNA in a polymerase chain reaction with a controltemplate DNA when used as a 3′ primer with an appropriate 5′ primer,such as SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence of an internal control scorpionprobe.

The sequences conform with 37 C.F.R. §§1.821-1.825 (“Requirements forPatent Applications Containing Nucleotide Sequences and/or Amino AcidSequence Disclosures—the Sequence Rules”) and are consistent with WorldIntellectual Property Organization (WIPO) Standard ST.25 (1998) and thesequence listing requirements of the EPO and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given as either a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

As used herein, the term “about” or “approximately” means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

The term “comprising” is intended to include embodiments encompassed bythe terms “consisting essentially of” and “consisting of”. Similarly,the term “consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”.

The term “pO157 portion” refers to the area of the E. coli O157:H7genome identified, for example, in Lukas M. Wick, et al., Evolution ofGenomic Content in the Stepwise Emergence of Escherichia coli O157:H7,Journal of Bacteriology 187:1783-91 (2005), as being divergent from theO55:H7 progenitor strain and as having been transferred into theprogenitor strain to create the O157:H7 E. coli serotype. This regionincludes, among other things, the O157-rfb gene cluster, colonic acidbiosynthesis genes, and putative type-1 fimbrial protein genes.

“Polymerase chain reaction” is abbreviated PCR.

The term “isolated” refers to materials, such as nucleic acid moleculesand/or proteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural, oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more strands of cDNA, genomic DNA,synthetic DNA, or mixtures thereof.

The term “amplification product” refers to nucleic acid fragmentsproduced during a primer-directed amplification reaction. Typicalmethods of primer-directed amplification include polymerase chainreaction (PCR), ligase chain reaction (LCR), or strand displacementamplification (SDA). If PCR methodology is selected, the replicationcomposition may comprise the components for nucleic acid replication,for example: nucleotide triphosphates, two (or more) primers withappropriate sequences, thermostable polymerase, buffers, solutes, andproteins. These reagents and details describing procedures for their usein amplifying nucleic acids are provided in U.S. Pat. No. 4,683,202(1987, Mullis, et al.) and U.S. Pat. No. 4,683,195 (1986, Mullis, etal.). If LCR methodology is selected, then the nucleic acid replicationcompositions may comprise, for example: a thermostable ligase (e.g.,Thermus aquaticus ligase), two sets of adjacent oligonucleotides(wherein one member of each set is complementary to each of the targetstrands), Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol, and salmonsperm DNA. See, for example, Tabor et al., Proc. Natl. Acad. Sci. U.S.A.82:1074-1078 (1985).

The term “primer” refers to an oligonucleotide (synthetic or occurringnaturally) that is capable of acting as a point of initiation of nucleicacid synthesis or replication along a complementary strand when placedunder conditions in which synthesis of a complementary strand iscatalyzed by a polymerase. A primer can further contain a detectablelabel, for example a 5′ end label.

The term “probe” refers to an oligonucleotide (synthetic or occurringnaturally) that is complementary (though not necessarily fullycomplementary) to a polynucleotide of interest and forms a duplexedstructure by hybridization with at least one strand of thepolynucleotide of interest. A probe or primer-probe complex can furthercontain a detectable label.

A probe can either be an independent entity or complexed with orotherwise attached to a primer, such as where a probe is connected viaits 3′ terminus to a primer's 5′ terminus through a linker, which may bea nucleotide or non-nucleotide linker and which may be a non-amplifiablelinker, such as a hexethylene glycol (HEG) or 18-carbon linker. In sucha case, this would be termed a “primer-probe complex.” One example ofsuch a primer-probe complex can be found in U.S. Pat. No. 6,326,145,incorporated herein by reference in its entirety, which are frequentlyreferred to as “Scorpion probes” or “Scorpion primers.”

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, semiconductor nanocrystals, ligands (e.g.,biotin, avidin, streptavidin, or haptens), and the like. A detectablelabel can also include a combination of a reporter and a quencher.

The term “reporter” refers to a substance or a portion thereof which iscapable of exhibiting a detectable signal, which signal can besuppressed by a quencher. The detectable signal of the reporter is,e.g., fluorescence in the detectable range. The term “quencher” refersto a substance or portion thereof which is capable of suppressing,reducing, inhibiting, etc., the detectable signal produced by thereporter.

As used herein, the terms “quenching” and “fluorescence energy transfer”refer to the process whereby, when a reporter and a quencher are inclose proximity, and the reporter is excited by an energy source, asubstantial portion of the energy of the excited state nonradiativelytransfers to the quencher where it either dissipates nonradiatively oris emitted at a different emission wavelength than that of the reporter.

Preferably, the reporter may be selected from fluorescent organic dyesmodified with a suitable linking group for attachment to theoligonucleotide, such as to the terminal 3′ carbon or terminal 5′carbon. The quencher may also be selected from organic dyes, which mayor may not be fluorescent, depending on the embodiment of the presentinvention. Generally, whether the quencher is fluorescent or simplyreleases the transferred energy from the reporter by non-radiativedecay, the absorption band of the quencher should at least substantiallyoverlap the fluorescent emission band of the reporter to optimize thequenching. Non-fluorescent quenchers or dark quenchers typicallyfunction by absorbing energy from excited reporters, but do not releasethe energy radiatively.

Selection of appropriate reporter-quencher pairs for particular probesmay be undertaken in accordance with known techniques. Fluorescent anddark quenchers and their relevant optical properties from whichexemplary reporter-quencher pairs may be selected are listed anddescribed, for example, in Berlman, Handbook of Fluorescence Spectra ofAromatic Molecules, 2nd ed., Academic Press, New York, 1971, the contentof which is incorporated herein by reference. Examples of modifyingreporters and quenchers for covalent attachment via common reactivegroups that can be added to an oligonucleotide in the present inventionmay be found, for example, in Haugland, Handbook of Fluorescent Probesand Research Chemicals, Molecular Probes of Eugene, Oreg., 1992, thecontent of which is incorporated herein by reference.

Preferred reporter-quencher pairs may be selected from xanthene dyesincluding fluoresceins and rhodamine dyes. Many suitable forms of thesecompounds are available commercially with substituents on the phenylgroups, which can be used as the site for bonding or as the bondingfunctionality for attachment to an oligonucleotide. Another preferredgroup of fluorescent compounds for use as reporters are thenaphthylamines, having an amino group in the alpha or beta position.Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5 sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include3-phenyl-7-isocyanatocoumarin; acridines such as9-isothiocyanatoacridine; N-(p-(2-benzoxazolyl)phenyl)maleimide;benzoxadiazoles; stilbenes; pyrenes and the like.

Most preferably, the reporters and quenchers are selected fromfluorescein and rhodamine dyes. These dyes and appropriate linkingmethodologies for attachment to oligonucleotides are well known in theart.

Suitable examples of quenchers may be selected from6-carboxy-tetramethylrhodamine, 4-(4-dimethylaminophenylazo)benzoic acid(DABYL), tetramethylrhodamine (TAMRA), BHQ-0™, BHQ-1™, BHQ-2™, andBHQ-3™, each of which are available from Biosearch Technologies, Inc. ofNovato, Calif., QSY-7™, QSY-9™, QSY-21™ and QSY-35™, each of which areavailable from Molecular Probes, Inc., and the like.

Suitable examples of reporters may be selected from dyes such as SYBRgreen, 5-carboxyfluorescein (5-FAM™ available from Applied Biosystems ofFoster City, Calif.), 6-carboxyfluorescein (6-FAM),tetrachloro-6-carboxyfluorescein (TET),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein,hexachloro-6-carboxyfluorescein (HEX),6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-TET™ available fromApplied Biosystems), carboxy-X-rhodamine (ROX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOE™ availablefrom Applied Biosystems), VIC™ dye products available from MolecularProbes, Inc., NED™ dye products available from available from AppliedBiosystems, and the like.

One example of a probe which contains a reporter and a quencher is aScorpion probe in either a unimolecular or bimolecular conformation. Ina unimolecular Scorpion, the probe portion of the primer-probe complexis flanked by self-complementary regions which allow the probe to forminto a stem-loop structure when the probe is unbound from its targetDNA. Examples of such self-complementary regions can be found in SEQ IDNO:4 and SEQ ID NO:15. Further, in a unimolecular Scorpion, a reporteris typically attached at or near one of the self-complementary regions,such as at the 5′ terminus of the Scorpion probe, and a quencher isattached at or near the other self-complementary region, such asimmediately 5′ to the non-amplifiable linker, such that the quencher isin sufficiently close proximity to the reporter to cause quenching whenthe probe is in its stem-loop conformation. In a bimolecular Scorpion,self-complementary flanking regions are not typically employed, butrather a separate “blocking oligonucleotide” is employed in conjunctionwith the Scorpion probe. This blocking oligonucleotide is capable ofhybridizing to the probe region of the Scorpion probe when the probe isunbound from its target DNA. An example of a bimolecular Scorpion pairis SEQ ID NO:10 (the Scorpion probe) and SEQ ID NO:11 (the blockingoligonucleotide). Further, in a bimolecular Scorpion, the reporter istypically attached to the probe region of the Scorpion probe, such as atthe 5′ terminus of the Scorpion probe, while the quencher is attached tothe blocking oligonucleotide, such as at the 3′ terminus of the blockingoligonucleotide, such that the quencher is in sufficiently closeproximity to the reporter to cause quenching when the probe is unboundfrom its target DNA and is instead hybridized to the blockingoligonucleotide.

Another example of a probe which contains a reporter and a quencher is aprobe that is to be used in a 5′-exonuclease assay, such as the Taqman®real-time PCR technique. In this context, the oligonucleotide probe willhave a sufficient number of phosphodiester linkages adjacent to its 5′end so that the 5′ to 3′ nuclease activity employed can efficientlydegrade the bound probe to separate the reporters and quenchers. Yetanother example of a probe which contains a reporter and quencher is aMolecular Beacon type probe, which contains a probe region flanked byself-complementary regions that allow the probe to form a stem-loopstructure when unbound from the probe's target sequence. Such probestypically have a reporter attached at or near one terminus and aquencher attached at or near the other terminus such that the quencheris in sufficiently close proximity to the reporter to cause quenchingwhen the probe is in its unbound, and thus stem-loop, form.

The term “replication inhibitor moiety” refers to any atom, molecule orchemical group that is attached to the 3′ terminal hydroxyl group of anoligonucleotide that will block the initiation of chain extension forreplication of a nucleic acid strand. Examples include, but are notlimited to: 3′-deoxynucleotides (e.g., cordycepin), dideoxynucleotides,phosphate, ligands (e.g., biotin and dinitrophenol), reporter molecules(e.g., fluorescein and rhodamine), carbon chains (e.g., propanol), amismatched nucleotide or polynucleotide, or peptide nucleic acid units.The term “non-participatory” refers to the lack of participation of aprobe or primer in a reaction for the amplification of a nucleic acidmolecule. Specifically a non-participatory probe or primer is one thatwill not serve as a substrate for, or be extended by, a DNA or RNApolymerase. A “non-participatory probe” is inherently incapable of beingchain extended by a polymerase. It may or may not have a replicationinhibitor moiety.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified, for example, in Sambrook, J., Fritsch, E. F. and Maniatis,T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to a Tm of55° C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringencyhybridization conditions correspond to a higher Tm, e.g., 40% formamide,with 5× or 6×SSC. Hybridization requires that the two nucleic acidscontain complementary sequences, although, depending on the stringencyof the hybridization, mismatches between bases are possible. Theappropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of Tm forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher Tm) of nucleic acid hybridizations decreases inthe following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greaterthan 100 nucleotides in length, equations for calculating Tm have beenderived (see Sambrook et al., supra, 9.50-9.51). For hybridizations withshorter nucleic acids, i.e., oligonucleotides, the position ofmismatches becomes more important, and the length of the oligonucleotidedetermines its specificity (see Sambrook et al., supra, 11.7-11.8). Inone preferred embodiment, the length for a hybridizable nucleic acid isat least about 10 nucleotides. More preferably a minimum length for ahybridizable nucleic acid is at least about 11 nucleotides, at leastabout 12 nucleotides, at least about 13 nucleotides, at least about 14nucleotides, at least about 15 nucleotides, at least about 16nucleotides, at least about 17 nucleotides, at least about 18nucleotides, at least about 19 nucleotides, at least about 20nucleotides, at least about 21 nucleotides, at least about 22nucleotides, at least about 23 nucleotides, at least about 24nucleotides, at least about 25 nucleotides, at least about 26nucleotides, at least about 27 nucleotides, at least about 28nucleotides, at least about 29 nucleotides, or, most preferably, atleast 30 nucleotides. Furthermore, the skilled artisan will recognizethat the temperature and wash solution salt concentration may beadjusted as necessary according to factors such as length of the probe.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by, e.g., Sambrook et al.(supra); and by Ausubel, F. M. et al., Current Protocols in MolecularBiology, published by Greene Publishing Assoc. and Wiley-Interscience(1987).

Genome Detection Regions

As discussed above, E. coli O157:H7 came about via the transfer of thepO157 plasmid into the O55:H7 progenitor strain. Thus, E. coli O157:H7possesses a pO157 portion within the bacterial genome. It has been foundthat the detection of a combination of regions both within and outsidethis pO157 portion of the E. coli O157:H7 genome produces a sensitiveand accurate method of detecting E. coli O157:H7, even in a backgroundof other serotypes of E. coli.

The present invention therefore relates to detection and identificationof E. coli O157:H7 through the detection of the presence of one or moreE. coli O157:H7 genomic DNA regions within the pO157 portion of thegenome in conjunction with the detection of the presence of one or moreE. coli O157:H7 genomic DNA regions outside the pO157 portion of thegenome. Preferably detection of E. coli O157:H7 is accomplished throughthe use of methods for detecting the presence of SEQ ID NO:1 inconjunction with one or more, more preferably both, of SEQ ID NOs: 2 and3. The present detection method finds utility in detection of E. coliO157:H7 in any type of sample, for example in appropriate samples forfood testing, environmental testing, or human or animal diagnostictesting. While examples of suitable methods for detecting these regionsare included herein, it is to be understood that the invention is notlimited to the methods described. Rather any suitable method can beemployed to detect these DNA regions and subsequently the E. coliitself.

Oligonucleotides

Oligonucleotides have been developed for the detection of the E. coliDNA regions SEQ ID NOs: 1-3 and the subsequent detection andidentification of E. coli serotype O157:H7. Oligonucleotides of theinstant invention are set forth in SEQ ID NOs: 4-20 and 24-26.

Oligonucleotides of the instant invention may be used as primers for PCRamplification. Preferred oligonucleotides for use as primers are SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:19, and SEQ ID NO:20. Particularly preferred primer pairs andtheir corresponding targets, blocking oligonucleotides, and probes areshown in Table 1.

TABLE 1 5′ (Forward) Blocking 3′ Target DNA Primer/Primer- Oligo-(Reverse) Region Probe Complex nucleotide Primer Probe SEQ ID SEQ ID N/ASEQ ID N/A NO: 1 NO: 4 NO: 5 SEQ ID SEQ ID N/A SEQ ID SEQ ID NO: 1 NO: 6NO: 5 NO: 7 SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: 2 NO: 10 NO: 11 NO: 12SEQ ID SEQ ID N/A SEQ ID SEQ ID NO: 2 NO: 13 NO: 12 NO: 14 SEQ ID SEQ IDN/A SEQ ID N/A NO: 3 NO: 15 NO: 16 SEQ ID SEQ ID N/A SEQ ID SEQ ID NO: 3NO: 17 NO: 16 NO: 18Each of these primers and probes was designed based on sequence analysisof its corresponding region of the E. coli O157:H7 genome. Primer designwas not aided by any software program.

These oligonucleotide primers may also be useful for other nucleic acidamplification methods such as the ligase chain reaction (LCR) (Backmanet al., 1989, EP 0 320 308; Carrino et al., 1995, J. Microbiol. Methods23: 3-20); nucleic acid sequence-based amplification (NASBA), (Carrinoet al., 1995, supra); and self-sustained sequence replication (3SR) and‘Q replicase amplification’ (Pfeffer et al., 1995 Veterinary Res. Comm.19: 375-407).

The oligonucleotide primers of the present invention can also contain adetectable label, for example a 5′ end label.

In addition, oligonucleotides of the present invention also may be usedas hybridization probes. Preferred hybridization probes are SEQ ID NO:4,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQID NO:24, SEQ ID NO:25, and SEQ ID NO:26. Hybridization using DNA probeshas been frequently used for the detection of pathogens in food,clinical and environmental samples, and the methodologies are generallyknown to one skilled in the art. It is generally recognized that thedegree of sensitivity and specificity of probe hybridization is lowerthan that achieved through the previously described amplificationtechniques. The nucleic acid probes of the present invention can alsopossess a detectable label, such as a reporter-quencher combination asare employed in Scorpion probe assays or in 5′-exonuclease detectionassays, such as the Taqman® assay.

The 3′ terminal nucleotide of the nucleic acid probe may be renderedincapable of extension by a nucleic acid polymerase in one embodiment ofthe invention. Such blocking may be carried out, for example by theattachment of a replication inhibitor moiety, such as a reporter orquencher, to the terminal 3′ carbon of the nucleic acid probe by alinking moiety, or by making the 3′-terminal nucleotide adideoxynucleotide. Alternatively, the 3′ end of the nucleic acid probemay be rendered impervious to the 3′ to 5′ extension activity of apolymerase by incorporating one or more modified internucleotidelinkages onto the 3′ end of the oligonucleotide. Minimally, the 3′terminal internucleotide linkage must be modified, however, additionalinternucleotide linkages may be modified. Internucleotide modificationswhich prevent elongation from the 3′ end of the nucleic acid probeand/or which block the 3′ to 5′ exonuclease activity of the DNApolymerase during PCR may include phosphorothioate linkages,methylphosphonate linkages, boranophosphate linkages, and other similarpolymerase-resistant internucleotide linkages. An alternative method toblock 3′ extension of the probe is to form an adduct at the 3′ end ofthe probe using mitomycin C or other like antitumor antibiotics such asdescribed in Basu et al., Biochemistry 32:4708-4718, 1993. Thus, theprecise mechanism by which the 3′ end of the nucleic acid probe isprotected from cleavage is not essential so long as the quencher is notcleaved from the nucleic acid probe.

A nucleic acid probe sequence can also optionally be employed with theprimer sequence pairs of the present invention in an amplification baseddetection technique, such as in the 3′-exonuclease assay. Preferredprimer/probe combinations are indicated in Table 1.

Some oligonucleotides of the present invention contain both primer andprobe regions, and thus can be employed as a primer-probe complex in anappropriate assay, such as a Scorpion probe assay. Examples of suchprimer-probe complexes of the current invention include SEQ ID NO:4, SEQID NO:10, and SEQ ID NO:15. These primer probe complexes of the instantinvention contain a non-amplifiable linker that connects the 3′ terminusof the probe region to the 5′ terminus of the primer region. Thisnon-amplifiable linker stops extension of a complementary strand fromproceeding into the probe region of the primer-probe complex. Examplesof such non-amplifiable linkages include hexethylene glycol (HEG) and,preferably, 18-carbon linkers. Primer-probe complexes of the presentinvention can also contain a self-complementary region that allows theprimer-probe complex to form a stem-loop structure when the probe isunbound from its target DNA, which may be useful, for example, inbringing the reporter and quencher into sufficiently close proximity toone another to cause the reporter signal to be quenched. Examples ofsuch primer-probe complexes with self-complementary regions include SEQID NO:4 and SEQ ID NO:15. Preferably, SEQ ID NO:4 is 5′ end-labeled witha Quasar670 reporter and also possesses a BHQ2 quencher at or near the3′ end of the probe region of this primer-probe complex (e.g., attachedto nucleotide 46). Preferably, SEQ ID NO:15 is 5′ end-labeled with aCalfluor Orange 560 reporter and also possesses a BHQ1 quencher at ornear the 3′ end of the probe region of this primer-probe complex (e.g.,attached to nucleotide 45). In some instances, a blockingoligonucleotide can be employed with a primer-probe complex, whichblocking oligonucleotide is capable of hybridizing to the probe regionof the primer-probe complex when the probe region is unbound from itstarget DNA. If the reporter is attached to the primer-probe complex andthe quencher is attached to the blocking oligonucleotide, this can bringthe reporter and quencher into sufficiently close proximity to oneanother to allow quenching to occur. For example, SEQ ID NO:11 is ablocking oligonucleotide capable of hybridizing to the primer-probecomplex SEQ ID NO:10. Preferably, SEQ ID NO:10 is 3′ end-labeled with a6FAM reporter while SEQ ID NO:11 is 5′ end-labeled with a BHQ1 quencher.

Assay Methods

Detection of the E. coli O157:H7 genomic DNA regions identified by SEQID NOs: 1-3, and subsequent detection of the presence of E. coli O157:H7itself, may be accomplished in any suitable manner. Preferred methodsare primer-directed amplification methods and nucleic acid hybridizationmethods. These methods may be used to detect E. coli O157:H7 in a samplethat is either a complex matrix or a purified culture, e.g., from ananimal, environmental, or food source suspected of contamination.

A preferred embodiment of the instant invention comprises (1) culturinga complex sample mixture in a non-selective growth media to resuscitatethe target bacteria, (2) releasing total target bacterial DNA, and (3)subjecting the total DNA to an amplification protocol with a primer pairof the invention and optionally with a nucleic acid probe comprising adetectable label.

Primer-Directed Amplification Assay Methods

A variety of primer-directed nucleic acid amplification methods areknown in the art which can be employed in the present invention,including thermal cycling methods (e.g., PCR, RT-PCR, and LCR), as wellas isothermal methods and strand displacement amplification (SDA). Thepreferred method is PCR. In one preferred embodiment, the primer pairslisted in Table 1 may be used as primers for use in primer-directednucleic acid amplification for the detection of SEQ ID NOs: 1-3 andsubsequently detection and identification of E. coli O157:H7.

Sample Preparation:

The oligonucleotides and methods according to the instant invention maybe used directly with any suitable clinical or environmental samples,without any need for sample preparation. In order to achieve highersensitivity, and in situations where time is not a limiting factor, itis preferred that the samples be pre-treated and that pre-amplificationenrichment is performed.

The minimum industry standard for the detection of food-borne bacterialpathogens is a method that will reliably detect the presence of onepathogen cell in 25 g of food matrix as described in Andrews et al.,1984, “Food Sample and Preparation of Sample Homogenate”, Chapter 1 inBacteriological Analytical Manual, 8th Edition, Revision A, Associationof Official Analytical Chemists, Arlington, Va. In order to satisfy thisstringent criterion, enrichment methods and media have been developed toenhance the growth of the target pathogen cell in order to facilitateits detection by biochemical, immunological or nucleic acidhybridization means. Typical enrichment procedures employ media thatwill enhance the growth and health of the target bacteria and alsoinhibit the growth of any background or non-target microorganismspresent. For example, the USDA has set forth a protocol for enrichmentof samples of ground beef to be tested for pathogenic E. coli (U.S. Foodand Drug Administration, Bacterial Analytical Manual).

Selective media have been developed for a variety of bacterial pathogensand one of skill in the art will know to select a medium appropriate forthe particular organism to be enriched, e.g. E. coli O157:H7. A generaldiscussion and recipes of non-selective media are described in the FDABacteriological Analytical Manual. (1998) published and distributed bythe Association of Analytical Chemists, Suite 400, 2200 Wilson Blvd,Arlington, Va. 22201-3301.

After selective growth, a sample of the complex mixtures is removed forfurther analysis. This sampling procedure may be accomplished by avariety of means well known to those skilled in the art. In a preferredembodiment, 5 μl of the enrichment culture is removed and added to 200μl of lysis solution containing protease. The lysis solution is heatedat 37° C. for 20 min followed by protease inactivation at 95° C. for 10min as described in the BAX® System User's Guide, DuPont Qualicon, Inc.,Wilmington, Del.

PCR Assay Methods:

A preferred method for detecting the presence of SEQ ID NOs: 1-3 andsubsequently E. coli O157:H7 in a sample comprises (a) performing PCRamplification of two or more of SEQ ID NOs: 1-3, preferably all three,using primer pairs listed in Table 1 to produce a PCR amplificationresult; and (b) detecting the amplification, whereby a positivedetection of the amplification indicates the presence of E. coli O157:H7in the sample.

In another preferred embodiment, prior to performing PCR amplification,a step of preparing the sample may be carried out. The preparing stepmay comprise at least one of the following processes: (1) bacterialenrichment, (2) separation of bacterial cells from the sample, (3) celllysis, and (4) total DNA extraction.

Amplification Conditions:

A skilled person will understand that any generally acceptable PCRconditions may be used for successfully detecting SEQ ID NOs: 1-3 andthe target E. coli O157:H7 bacteria using the oligonucleotides of theinstant invention, and depending on the sample to be tested and otherlaboratory conditions, routine optimization for the PCR conditions maybe necessary to achieve optimal sensitivity and specificity. Optimally,they achieve PCR amplification results from all of the intended specifictargets while giving no PCR results for other, non-target species.

Detection/Examination/Analysis:

Primer-directed amplification products of SEQ ID NOs: 1-3 can beanalyzed using various methods. Homogenous detection refers to apreferred method for the detection of amplification products where noseparation (such as by gel electrophoresis) of amplification productsfrom template or primers is necessary. Homogeneous detection istypically accomplished by measuring the level of fluorescence of thereaction mixture during or immediately following amplification. Inaddition, heterogeneous detection methods, which involve separation ofamplification products during or prior to detection, can be employed inthe present invention.

Homogenous detection may be employed to carry out “real-time”primer-directed nucleic acid amplification and detection, using primerpairs of the instant invention (e.g., “real-time” PCR and “real-time”RT-PCR). Preferred “real-time” methods are set forth in U.S. Pat. Nos.6,171,785, 5,994,056, 6,326,145, 5,804,375, 5,538,848, 5,487,972, and5,210,015, each of which is hereby incorporated by reference in itsentirety.

A particularly preferred “real-time” detection method is the Scorpionprobe assay as set forth in U.S. Pat. No. 6,326,145, which is herebyincorporated by reference in its entirety. In the Scorpion probe assay,PCR amplification is performed using a Scorpion probe (eitherunimolecular or bimolecular) as a primer-probe complex, the Scorpionprobe possessing an appropriate reporter-quencher pair to allow thedetectable signal of the reporter to be quenched prior to elongation ofthe primer. Post-elongation, the quenching effect is eliminated and theamount of signal present is quantitated. As the amount of amplificationproduct increases, an equivalent increase in detectable signal will beobserved, thus allowing the amount of amplification product present tobe determined as a function of the amount of detectable signal measured.When more than one Scorpion probe is employed in a Scorpion probe assayof present invention, such as one directed to each of the three DNAregions of interest (SEQ ID NOs: 1-3), each probe can have a differentdetectable label (e.g., reporter-quencher pair) attached, thus allowingeach probe to be detected independently of the other probes.

In a preferred embodiment of the present invention, amplification anddetection of all three of SEQ ID NOs: 1-3 is performed usingdifferentially labeled Scorpion probes. SEQ ID NO:1 is preferablyamplified and detected using SEQ ID NO:4 in conjunction with SEQ IDNO:5, with SEQ ID NO:4 possessing a Quasar 670 reporter attached at the5′ terminus and a BHQ2 quencher attached immediately upstream (i.e., inthe 5′ direction) of the non-amplifiable linker, preferably an 18-carbonlinker. SEQ ID NO:2 is preferably amplified and detected using SEQ IDNO:10 in conjunction with SEQ ID NO:11 and SEQ ID NO:12, with SEQ IDNO:10 possessing a 6FAM reporter attached at the 5′ terminus and SEQ IDNO:11 possessing a BHQ1 quencher attached at its 3′ terminus. SEQ IDNO:3 is preferably amplified and detected using SEQ ID NO:15 inconjunction with SEQ ID NO:16, with SEQ ID NO:15 possessing a CalfluorOrange 560 reporter attached at the 5′ terminus and a BHQ1 quencherattached immediately upstream (i.e., in the 5′ direction) of thenon-amplifiable linker, preferably an 18-carbon linker.

Another preferred “real-time” detection method is the 5′-exonucleasedetection method, as set forth in U.S. Pat. Nos. 5,804,375, 5,538,848,5,487,972, and 5,210,015, each of which is hereby incorporated byreference in its entirety. In the 5′-exonuclease detection assay amodified probe is employed during PCR which binds intermediate to orbetween the two members of the amplification primer pair. The modifiedprobe possesses a reporter and a quencher and is designed to generate adetectable signal to indicate that it has hybridized with the targetnucleic acid sequence during PCR. As long as both the reporter and thequencher are on the probe, the quencher stops the reporter from emittinga detectable signal. However, as the polymerase extends the primerduring amplification, the intrinsic 5′ to 3′ nuclease activity of thepolymerase degrades the probe, separating the reporter from thequencher, and enabling the detectable signal to be emitted. Generally,the amount of detectable signal generated during the amplification cycleis proportional to the amount of product generated in each cycle.

It is well known that the efficiency of quenching is a strong functionof the proximity of the reporter and the quencher, i.e., as the twomolecules get closer, the quenching efficiency increases. As quenchingis strongly dependent on the physical proximity of the reporter andquencher, the reporter and the quencher are preferably attached to theprobe within a few nucleotides of one another, usually within 30nucleotides of one another, more preferably with a separation of fromabout 6 to 16 nucleotides. Typically, this separation is achieved byattaching one member of a reporter-quencher pair to the 5′ end of theprobe and the other member to a nucleotide about 6 to 16 nucleotidesaway.

Again, when more than one Taqman® probe is employed in a 5′-exonucleasedetection assay of present invention, such as one directed to each ofthe three DNA regions of interest (SEQ ID NOs: 1-3), each probe can havea different detectable label (e.g., reporter-quencher pair) attached,thus allowing each probe to be detected independently of the otherprobes. Preferred Taqman® probes of the present invention include SEQ IDNOs:24-26, which detect SEQ ID NO:2, SEQ ID NO:1, and SEQ ID NO:3,respectively.

Another preferred method of homogenous detection involves the use of DNAmelting curve analysis, particularly with the BAX® System hardware andreagent tablets from DuPont Qualicon Inc. The details of the system aregiven in U.S. Pat. No. 6,312,930 and PCT Publication Nos. WO 97/11197and WO 00/66777, each of which is hereby incorporated by reference inits entirety.

Melting curve analysis detects and quantifies double stranded nucleicacid molecule (“dsDNA” or “target”) by monitoring the fluorescence ofthe target amplification product (“target amplicon”) during eachamplification cycle at selected time points.

As is well known to the skilled artisan, the two strands of a dsDNAseparate or melt, when the temperature is higher than its meltingtemperature. Melting of a dsDNA molecule is a process, and under a givensolution condition, melting starts at a temperature (designated T_(MS)hereinafter), and completes at another temperature (designated T_(ME)hereinafter). The familiar term, T_(m), designates the temperature atwhich melting is 50% complete.

A typical PCR cycle involves a denaturing phase where the target dsDNAis melted, a primer annealing phase where the temperature optimal forthe primers to bind to the now-single-stranded target, and a chainelongation phase (at a temperature T_(E)) where the temperature isoptimal for DNA polymerase to function.

According to the present invention, T_(MS) should be higher than T_(E),and T_(ME) should be lower (often substantially lower) than thetemperature at which the DNA polymerase is heat-inactivated. Meltingcharacteristics are effected by the intrinsic properties of a givendsDNA molecule, such as deoxynucleotide composition and the length ofthe dsDNA.

Intercalating dyes will bind to double stranded DNA. The dye/dsDNAcomplex will fluoresce when exposed to the appropriate excitationwavelength of light, which is dye dependent, and the intensity of thefluorescence may be proportionate to concentration of the dsDNA. Methodstaking advantage of the use of DNA intercalating dyes to detect andquantify dsDNA are known in the art. Many dyes are known and used in theart for these purposes. The instant methods also take advantage of suchrelationship.

Examples of such intercalating dyes include, but are not limited to,SYBR Green-I®, ethidium bromide, propidium iodide, TOTO®-1 {Quinolinium,1-1′-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]]-,tetraiodide},and YoPro® {Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)-propyl]-,diiodide}.Most preferred for the instant invention is a non-asymmetrical cyanidedye such as SYBR Green-I®, manufactured by Molecular Probes, Inc.(Eugene, Oreg.).

Melting curve analysis is achieved by monitoring the change influorescence while the temperature is increased. When the temperaturereaches the T_(MS) specific for the target amplicon, the dsDNA begins todenature. When the dsDNA denatures, the intercalating dye dissociatesfrom the DNA and fluorescence decreases. Mathematical analysis of thenegative of the change of the log of fluorescence divided by the changein temperature plotted against the temperature results in the graphicalpeak known as a melting curve.

It should be understood that the present invention could be operatedusing a combination of these techniques, such as by having a Scorpionprobe directed to one target region and a Taqman® probe directed to asecond target region. It should also be understood that the invention isnot limited to the above described techniques. Rather, one skilled inthe art would recognize that other techniques for detectingamplification as known in the art may also be used. For example,techniques such as PCR-based quantitative sequence detection (QSD) maybe performed using nucleic acid probes which, when present in thesingle-stranded state in solution, are configured such that the reporterand quencher are sufficiently close to substantially quench thereporter's emission. However, upon hybridization of the intactreporter-quencher nucleic acid probe with the amplified target nucleicacid sequence, the reporter and quenchers become sufficiently distantfrom each other. As a result, the quenching is substantially abatedcausing an increase in the fluorescence emission detected.

In addition to homogenous detection methods, a variety of otherheterogeneous detection methods are known in the art which can beemployed in the present invention, including standard non-denaturing gelelectrophoresis (e.g., acrylamide or agarose), denaturing gradient gelelectrophoresis, and temperature gradient gel electrophoresis. Standardnon-denaturing gel electrophoresis is a simple and quick method of PCRdetection, but may not be suitable for all applications.

Denaturing Gradient Gel Electrophoresis (DGGE) is a separation methodthat detects differences in the denaturing behavior of small DNAfragments (200-700 bp). The principle of the separation is based on bothfragment length and nucleotide sequence. In fragments that are the samelength, a difference as little as one base pair can be detected. This isin contrast to non-denaturing gel electrophoresis, where DNA fragmentsare separated only by size. This limitation of non-denaturing gelelectrophoresis results because the difference in charge density betweenDNA molecules is near neutral and plays little role in their separation.As the size of the DNA fragment increases, its velocity through the geldecreases.

DGGE is primarily used to separate DNA fragments of the same size basedon their denaturing profiles and sequence. Using DGGE, two strands of aDNA molecule separate, or melt, when heat or a chemical denaturant isapplied. The denaturation of a DNA duplex is influenced by twofactors: 1) the hydrogen bonds formed between complimentary base pairs(since GC rich regions melt at higher denaturing conditions than regionsthat are AT rich); and 2) the attraction between neighboring bases ofthe same strand, or “stacking.” Consequently, a DNA molecule may haveseveral melting domains with each of their individual characteristicdenaturing conditions determined by their nucleotide sequence. DGGEexploits the fact that otherwise identical DNA molecules having the samelength and DNA sequence, with the exception of only one nucleotidewithin a specific denaturing domain, will denature at differenttemperatures or Tm. Thus, when the double-stranded (ds) DNA fragment iselectrophoresed through a gradient of increasing chemical denaturant itbegins to denature and undergoes both a conformational and mobilitychange. The dsDNA fragment will travel faster than a denaturedsingle-stranded (ss) DNA fragment, since the branched structure of thesingle-stranded moiety of the molecule becomes entangled in the gelmatrix. As the denaturing environment increases, the dsDNA fragment willcompletely dissociate and mobility of the molecule through the gel isretarded at the denaturant concentration at which the particular lowdenaturing domains of the DNA strand dissociate. In practice, theelectrophoresis is conducted at a constant temperature (around 60° C.)and chemical denaturants are used at concentrations that will result in100% of the DNA molecules being denatured (i.e., 40% formamide and 7Murea). This variable denaturing gradient is created using a gradientmaker, such that the composition of each DGGE gel gradually changes from0% denaturant up to 100% denaturant. Of course, gradients containing areduced range of denaturant (e.g., 35% to 60%) may also be poured forincreased separation of DNA.

The principle used in DGGE can also be applied to a second method thatuses a temperature gradient instead of a chemical denaturant gradient.This method is known as Temperature Gradient Gel Electrophoresis (TGGE).This method makes use of a temperature gradient to induce theconformational change of dsDNA to ssDNA to separate fragments of equalsize with different sequences. As in DGGE, DNA fragments with differentnucleotide sequences will become immobile at different positions in thegel. Variations in primer design can be used to advantage in increasingthe usefulness of DGGE for characterization and identification of thePCR products. These methods and principles of using primer designvariations are described in PCR Technology Principles and Applications,Henry A. Erlich Ed., M. Stockton Press, NY, pages 71 to 88 (1988).

Instrumentation:

When homogenous detection is employed, the level of fluorescence ispreferably measured using a laser fluorometer such as, for example, anABI Prism Model 7500 Fast Sequence Detector. However, similar detectionsystems for measuring the level of fluorescence in a sample are includedin the invention.

Reagents and Kits:

Any suitable nucleic acid replication composition (“replicationcomposition”) in any format can be used. A typical replicationcomposition for PCR amplification may comprise, for example, dATP, dCTP,dGTP, dTTP, target specific primers and a suitable polymerase.

If the replication composition is in liquid form, suitable buffers knownin the art may be used (Sambrook, J. et al., supra).

Alternatively, if the replication composition is contained in a tabletform, then typical tabletization reagents may be included such asstabilizers and binding agents. Preferred tabletization technology isset forth in U.S. Pat. Nos. 4,762,857 and 4,678,812, each of which ishereby incorporated by reference in its entirety.

A preferred replication composition of the instant invention comprises(a) at least one primer pair selected from Table 1, and (b) thermostableDNA polymerase. Another preferred replication composition comprises (a)at least two primer pairs selected from Table 1, each directed toward adifferent target DNA region; and (b) thermostable DNA polymerase. Morepreferred is inclusion of primer pairs directed to all three of SEQ IDNOs: 1-3.

A more preferred replication composition of the present inventioncomprises (a) at least two primer pairs and any corresponding probe orblocking oligonucleotide selected from Table 1, wherein each nucleicacid probe or primer-probe complex employed comprises a detectablelabel; and (b) thermostable DNA polymerase. Preferably the detectablelabel comprises a reporter capable of emitting a detectable signal and aquencher capable of substantially quenching the reporter and preventingthe emission of the detectable signal when the reporter and quencher arein sufficiently close proximity to one another.

A preferred kit of the instant invention comprises any one of the abovereplication compositions. A preferred tablet of the instant inventioncomprises any one of the above replication compositions. Morepreferably, a kit of the instant invention comprises the foregoingpreferred tablet.

In some instances, an internal positive control can be included in thereaction. The internal positive control can include control templatenucleic acids (e.g. DNA or RNA), control primers, and control nucleicacid probe. The advantages of an internal positive control containedwithin a PCR reaction have been previously described (U.S. Pat. No.6,312,930 and PCT Application No. WO 97/11197, each of which is herebyincorporated by reference in its entirety), and include: (i) the controlmay be amplified using a single primer; (ii) the amount of the controlamplification product is independent of any target DNA or RNA containedin the sample; (iii) the control DNA can be tableted with otheramplification reagents for ease of use and high degree ofreproducibility in both manual and automated test procedures; (iv) thecontrol can be used with homogeneous detection, i.e., without separationof product DNA from reactants; and (v) the internal control has amelting profile that is distinct from other potential amplificationproducts in the reaction and/or a detectable label on the controlnucleic acid that is distinct from the detectable label on the nucleicacid probe directed to the target.

Control DNA will be of appropriate size and base composition to permitamplification in a primer-directed amplification reaction. The controltemplate DNA sequence may be obtained from the E. coli genome, or fromanother source, but must be reproducibly amplified under the sameconditions that permit the amplification of the target amplificationproduct.

Preferred control sequences include, for example, control primers SV4219(SEQ ID NO:21) and SV4313 (SEQ ID NO:22) and probe SV40Probe3 (SEQ IDNO:23) for Taqman® assays and control primers SV40-33-4312 (SEQ IDNO:27) and SV40-29-4222 (SEQ ID NO:28) and control probe SV40 scorpion 1(SEQ ID NO:29) for Scorpion assays.

The control reaction is useful to validate the amplification reaction.Amplification of the control DNA occurs within the same reaction tube asthe sample that is being tested, and therefore indicates a successfulamplification reaction when samples are target negative, i.e. no targetamplification product is produced. In order to achieve significantvalidation of the amplification reaction, a suitable number of copies ofthe control DNA template must be included in each amplificationreaction.

In some instances it may be useful to include an additional negativecontrol replication composition. The negative control replicationcomposition will contain the same reagents as the replicationcomposition but without the polymerase. The primary function of such acontrol is to monitor spurious background fluorescence in a homogeneousformat when the method employs a fluorescent means of detection.

Replication compositions may be modified depending on whether they aredesigned to be used to amplify target DNA or the control DNA.Replication compositions that will amplify the target DNA (testreplication compositions) may include (i) a polymerase (generallythermostable), (ii) a primer pair capable of hybridizing to the targetDNA and (iii) necessary buffers for the amplification reaction toproceed. Replication compositions that will amplify the control DNA(positive control, or positive replication composition) may include (i)a polymerase (generally thermostable) (ii) the control DNA; (iii) atleast one primer capable of hybridizing to the control DNA; and (iv)necessary buffers for the amplification reaction to proceed. Inaddition, the replication composition for either target DNA or controlDNA amplification can contain a nucleic acid probe, preferablypossessing a detectable label.

Nucleic Acid Hybridization Methods

In addition to primer-directed amplification assay methods, nucleic acidhybridization assay methods can be employed in the present invention fordetection of E. coli O157:H7. The basic components of a nucleic acidhybridization test include a probe, a sample suspected of containing E.coli O157:H7, and a specific hybridization method. Typically the probelength can vary from as few as 5 bases to the full length of the E. colidiagnostic sequence and will depend upon the specific test to be done.Only part of the probe molecule need be complementary to the nucleicacid sequence to be detected. In addition, the complementarity betweenthe probe and the target sequence need not be perfect. Hybridizationdoes occur between imperfectly complementary molecules with the resultthat a certain fraction of the bases in the hybridized region are notpaired with the proper complementary base.

Probes particularly useful in nucleic acid hybridization methods are anyof SEQ ID NOs:4-20 or 24-26, or sequences derived therefrom.

The sample may or may not contain E. coli O157:H7. The sample may take avariety of forms, however will generally be extracted from an animal,environmental or food source suspected of contamination. The DNA may bedetected directly but most preferably, the sample nucleic acid must bemade available to contact the probe before any hybridization of probeand target molecule can occur. Thus the organism's DNA is preferablyfree from the cell and placed under the proper conditions beforehybridization can occur. Methods of in-solution hybridizationnecessitate the purification of the DNA in order to be able to obtainhybridization of the sample DNA with the probe. This has meant thatutilization of the in-solution method for detection of target sequencesin a sample requires that the nucleic acids of the sample must first bepurified to eliminate protein, lipids, and other cell components, andthen contacted with the probe under hybridization conditions. Methodsfor the purification of the sample nucleic acid are common and wellknown in the art (Sambrook et al., supra).

In one preferred embodiment, hybridization assays may be conducteddirectly on cell lysates, without the need to extract the nucleic acids.This eliminates several steps from the sample-handling process andspeeds up the assay. To perform such assays on crude cell lysates, achaotropic agent is typically added to the cell lysates prepared asdescribed above. The chaotropic agent stabilizes nucleic acids byinhibiting nuclease activity. Furthermore, the chaotropic agent allowssensitive and stringent hybridization of short oligonucleotide probes toDNA at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151 (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide, and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Alternatively, one can purify the sample nucleic acids prior to probehybridization. A variety of methods are known to one of skill in the art(e.g., phenol-chloroform extraction, Isoquick extraction (MicroProbeCorp., Bothell, Wash.), and others). Pre-hybridization purification isparticularly useful for standard filter hybridization assays.Furthermore, purification facilitates measures to increase the assaysensitivity by incorporating in vitro RNA amplification methods such asself-sustained sequence replication (see for example Fahy et al., In PCRMethods and Applications, Cold Spring Harbor Laboratory: Cold SpringHarbor, N.Y. (1991), pp. 25-33) or reverse transcriptase PCR (Kawasaki,In PCR Protocols: A Guide to Methods and Applications, M. A. Innis etal., Eds., (1990), pp. 21-27).

Once the DNA is released, it can be detected by any of a variety ofmethods. However, the most useful embodiments have at least somecharacteristics of speed, convenience, sensitivity, and specificity.

Hybridization methods are well known in the art. Typically the probe andsample must be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed.

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers,such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9),about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents which include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate), and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the DNA sequence.

The sandwich assay may be encompassed in an assay kit. This kit wouldinclude a first component for the collection of samples suspected ofcontamination and buffers for the disbursement and lysis of the sample.A second component would include media in either dry or liquid form forthe hybridization of target and probe polynucleotides, as well as forthe removal of undesirable and nonduplexed forms by washing. A thirdcomponent includes a solid support (dipstick) upon which is fixed (or towhich is conjugated) unlabeled nucleic acid probe(s) that is (are)complementary to one or more of SEQ ID NOs: 1-3. A fourth componentwould contain labeled probe that is complementary to a second anddifferent region of the same DNA strand to which the immobilized,unlabeled nucleic acid probe of the third component is hybridized.

In a preferred embodiment, SEQ ID NOs: 4-20 or derivations thereof maybe used as 3′ blocked detection probes in either a homogeneous orheterogeneous assay format. For example, a probe generated from thesesequences may be 3′ blocked or non-participatory and will not beextended by, or participate in, a nucleic acid amplification reaction.Additionally, the probe incorporates a label that can serve as areactive ligand that acts as a point of attachment for theimmobilization of the probe/analyte hybrid or as a reporter to producedetectable signal. Accordingly, genomic or cDNA isolated from a samplesuspected of E. coli contamination is amplified by standardprimer-directed amplification protocols in the presence of an excess ofthe 3′ blocked detection probe to produce amplification products.Because the probe is 3′ blocked, it does not participate or interferewith the amplification of the target. After the final amplificationcycle, the detection probe anneals to the relevant portion of theamplified DNA and the annealed complex is then captured on a supportthrough the reactive ligand.

In some instances it is desirable to incorporate a ligand labeled dNTP,with the label probe in the replication composition to facilitateimmobilization of the PCR reaction product on a support and thendetection of the immobilized product by means of the labeled probereagent. For example a biotin, digoxigenin, or digoxin labeled dNTPcould be added to PCR reaction composition. The biotin, digoxigenin, ordigoxin incorporated in the PCR product could then be immobilizedrespectively on to a strepavidin, anti-dixogin or antidigoxigeninantibody support. The immobilized PCR product could then be detected bythe presence of the probe label.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.

General Methods and Materials Used in the Examples

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found in Manual of Methods forGenus Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds), American Society for Microbiology, Washington, D.C.(1994) or Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass. or Bacteriological Analytical Manual. 6th Edition,Association of Official Analytical Chemists, Arlington, Va. (1984).

The medium used to grow the pathogenic E. coli strains and comparativenon-target strains was Brain Heart Infusion broth (BHI) obtained fromBBL (Becton-Dickenson). Samples of pathogenic E. coli strains wereobtained from cultures grown overnight in BHI broth then diluted toapproximately 10⁶ cfu/ml in 0.1% peptone water. Samples of thecomparative non-target strains were enriched in BHI at approximately 10⁹cfu/ml.

Primers and probes (SEQ ID NOs: 4-29) were prepared by Sigma-Genosys,Woodlands, Tex.

All PCR reactions were carried out using a standard BAX® System (DuPontQualicon, Wilmington, Del.).

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “ml” meansmilliliter(s), “μl” means microliter(s), “cfu” means colony formingunit(s).

Example 1 Determination of Inclusivity/Exclusivity of the IndividualTargets Via Taqman® Assay

Samples of organisms were analyzed to establish inclusivity andexclusivity of individual Taqman® probes of the present invention. Purecultures grown overnight achieved cell densities of 8×10⁸ to 2×10⁹cfu/ml. For inclusivity, independent, bona fide E. coli O157:H7 isolateswere used; for exclusivity non O157:H7 E. coli were used to ensure thatthe assay would discriminate the target organism (O157:H7) from other E.coli.

DNA Lysate Preparation

Material tested was either food enrichment (ground beef enrichmentprepared as described in the BAX® system user guide for the BAX® MPassay) or overnight growth of E. coli O157:H7 isolates at 37° C. in BHImedia. 20 μl of the material to be tested was added to 200 μl of BAX®lysis reagent (DuPont Qualicon, Wilmington, Del.). The mixture wasincubated at 37° C. for 20 minutes, then further incubated at 95° C. for10 minutes, and finally cooled to 5° C.

PCR Conditions

30 μl of the DNA lysate as prepared above was used to hydratelyophylized PCR reaction components to achieve a DNA lysate/PCR reactioncomponent mixture containing the primers and probes listed in TABLE 2.

TABLE 2 Quench- Per reaction 5′ Fluor er Primers wbdRa1F 15 pMole (SEQID NO: 17) wbdra2R 15 pMole (SEQ ID NO: 16) PerSt73F 12 pMole (SEQ IDNO: 13) PerSt73R 12 pMole (SEQ ID NO: 12) SIS1T74F 12 pMole (SEQ ID NO:6) SIS1T75R 12 pMole (SEQ ID NO: 5) SV4219 20 pMole (SEQ ID NO: 21)SV4313 20 pMole (SEQ ID NO: 22) Probes SV40Probe3 10 pMole Calflour BHQ1(SEQ ID NO: 23) Orange 560 Perstalt166p  5 pMole Quasar 670 BHQ1 (SEQ IDNO: 24) SIS1T65P 10 pMole FAM BHQ1 (SEQ ID NO: 25) wbdRa1P 12.5 pMole Tamra BHQ1 (SEQ ID NO: 26)This DNA lysate/PCR reaction component mixture was added to a PCRreaction mixture. The reagents that were used in the PCR amplificationreaction were from BAX® System Reagent Tablet Kits (DuPont Qualicon,Wilmington, Del.) and include SYBR® Green (Molecular Probes, Eugene,Oreg.), Taq DNA Polymerase (Applied Biosystems, Foster City, Calif.),deoxynucleotides (Roche Diagnostics, Indianapolis, Ind.), and buffer (EMScience, New Jersey).

Amplification and testing was performed on the BAX® Q7 machine (DuPontQualicon, Wilmington, Del.). The thermal cycling conditions were: 2minutes at 94° C., followed by 40 cycles of 94° C. for 10 seconds and60° C. for 30 seconds, with the fluorescent signal captured during the60° C. step at each cycle.

Results

As can be seen in Table 3, below, using individual Taqman® probes, themethod of the present invention was able to almost completelydistinguish between O157:H7 and non-O157:H7 E. coli strains.

TABLE 3 Inclusivity Panel: 36 E. coli O157:H7 isolates Taqman ® VersionPerosamine SIS1 Synthetase wbdr Sample Target 1 Target 2 Target 3 IDResult Result Result DD935 Positive Positive Positive DD1449 PositivePositive Positive DD1450 Positive Positive Positive DD1451 PositivePositive Positive DD1452 Positive Positive Positive DD1453 PositivePositive Positive DD1454 Positive Positive Positive DD1455 PositivePositive Positive DD1456 Positive Positive Positive DD1457 PositivePositive Positive DD1458 Positive Positive Positive DD1459 PositivePositive Positive DD1460 Positive Positive Positive DD1461 PositivePositive Positive DD1462 Positive Positive Positive DD1463 PositivePositive Positive DD1972 Positive Positive Positive DD1973 PositivePositive Positive DD1974 Positive Positive Positive DD1975 PositivePositive Positive DD1976 Positive Positive Positive DD1977 PositivePositive Positive DD1978 Positive Positive Positive DD1979 PositivePositive Positive DD1980 Positive Positive Positive DD1981 PositivePositive Positive DD1982 Positive Positive Positive DD1983 PositivePositive Positive DD1984 Positive Positive Positive DD1985 PositivePositive Positive DD1986 Positive Positive Positive DD1987 PositivePositive Positive DD1988 Positive Positive Positive DD1989 PositivePositive Positive DD1990 Positive Positive Positive DD1991 PositivePositive Positive

TABLE 4 Exclusivity Panel: 45 E. coli non-O157:H7 isolates Taqman ®Version Perosamine SIS1 Synthetase wbdr Sample Target 1 Target 2 Target3 ID Result Result Result DD1797 Negative — — DD1808 Negative NegativeNegative DD1809 Negative Negative Negative DD1821 Negative NegativeNegative DD1845 Negative Negative Negative DD1858 Negative NegativeNegative DD1859 Negative Negative Negative DD1869 Negative NegativeNegative DD1870 Negative Negative Negative DD1906 Negative NegativeNegative DD1915 Negative Negative Negative DD1927 Negative NegativeNegative DD1931 Negative Negative Negative DD2448 Negative NegativeNegative DD2450 Negative Negative Negative DD2453 Negative NegativeNegative DD2474 Negative Negative Negative DD2508 Negative NegativeNegative DD2511 Negative Negative Negative DD2514 Negative NegativeNegative DD2517 Negative Negative Negative DD2518 Negative NegativeNegative DD2522 Negative Negative Negative DD2523 Negative NegativeNegative DD3124 Negative Negative Negative DD3127 Negative NegativeNegative DD3130 Negative Negative Negative DD3132 Negative NegativeNegative DD3166 Negative Negative Negative DD3197 Negative NegativeNegative DD3199 Negative Negative Negative DD3204 Negative NegativeNegative DD3208 Negative Negative Negative DD3210 Negative NegativeNegative DD3785 Negative Negative Negative DD5884 Negative NegativeNegative DD5887 Negative Negative Negative DD5901 Negative NegativeNegative DD9705 Negative Negative Negative DD10910 Positive NegativeNegative DD12901 Negative Negative Negative DD5883 Negative NegativeNegative DD12800 Negative Negative Negative DD12804 Negative NegativeNegative DD12849 Negative Negative Negative DD12851 Negative NegativeNegative

Example 2 Determination of Inclusivity/Exclusivity of the IndividualTargets Via Scorpion Assay

Samples of organisms were analyzed to establish inclusivity andexclusivity of individual Scorpion probes of the present invention. Purecultures grown overnight achieved cell densities of 8×10⁸ to 2×10⁹cfu/ml. For inclusivity, independent, bona fide E. coli O157:H7 isolateswere used; for exclusivity non O157:H7 E. coli were used to ensure thatthe assay would discriminate the target organism (O157:H7) from other E.coli.

DNA Lysate Preparation

Material tested was either food enrichment (ground beef enrichmentprepared as described in the BAX® system user guide for the BAX® MPassay) or overnight growth of E. coli O157:H7 isolates at 37° C. in BHImedia. 20 μl of the material to be tested was added to 200 μl of BAX®lysis reagent (DuPont Qualicon, Wilmington, Del.). The mixture wasincubated at 37° C. for 20 minutes, then further incubated at 95° C. for10 minutes, and finally cooled to 5° C.

PCR Conditions

30 μl of the DNA lysate as prepared above was used to hydratelyophilized PCR reaction components to achieve a DNA lysate/PCR reactioncomponent mixture containing the primers and probes listed in TABLE 5.

TABLE 5 Quench- Per reaction 5′ Fluor er Primers wbdra2R 20 pMole (SEQID NO: 16) PerSt73R 20 pMole (SEQ ID NO: 12) SIS1T75R 20 pMole (SEQ IDNO: 5) SV40-33-4312 20 pMole (SEQ ID NO: 27) SV40-29-4222 2.5 pMole (SEQ ID NO: 28) Probes WBDR scorpion 1 15 pMole Calflour BHQ1 (SEQ IDNO: 15) Orange 560 SIS1 scorpion 1  5 pMole Quasar 670 BHQ2 (SEQ ID NO:4) SV40 scorpion 1 10 pMole Tamra BHQ2 (SEQ ID NO: 29) PERSYNA probe 120 pMole 6FAM n/a (SEQ ID NO: 10) PERSYNA probe 2 200 pMole  n/a BHQ1(SEQ ID NO: 11) n/a = not applicableSEQ ID NO:15 (WBDR scorpion 1 probe) is presented in the sequencelisting as the entire sequence without modifications. For this Example,the WBDR scorpion probe has, following nucleotide number 45 in SEQ IDNO:15, an internal BHQ1 quencher and an SP-18 blocker followed by theremaining 42 nucleotides. Similarly, SEQ ID NO:4 (SIS1 scorpion 1 probe)is presented in the sequence listing as the entire sequence withoutmodifications. For this Example, the SIS1 scorpion probe has, followingnucleotide number 46 in SEQ ID NO:4, an internal BHQ2 quencher and anSP-18 blocker followed by the remaining 40 nucleotides. Also, SEQ IDNO:29 (SV40 scorpion 1 probe) is presented in the sequence listing asthe entire sequence without modifications. For this Example, the SV40scorpion probe has, following nucleotide number 52 in SEQ ID NO:29, aninternal BHQ2 quencher and an SP-18 blocker followed by the remaining 26nucleotides.

The DNA lysate/PCR reaction component mixture was added to a PCRreaction mixture. The reagents that were used in the PCR amplificationreaction were from BAX® System Reagent Tablet Kits (DuPont Qualicon,Wilmington, Del.) and include SYBR® Green (Molecular Probes, Eugene,Oreg.), Taq DNA Polymerase (Applied Biosystems, Foster City, Calif.),deoxynucleotides (Roche Diagnostics, Indianapolis, Ind.), and buffer (EMScience, New Jersey).

Amplification and testing was performed on the BAX® Q7 machine (DuPontQualicon, Wilmington, Del.). The thermal cycling conditions were: 2minutes at 94° C., followed by 40 cycles of 94° C. for 10 seconds and63° C. for 40 seconds, with the fluorescent signal captured during the63° C. step at each cycle.

Results

As can be seen in Table 6, below, using individual Scorpion probes, themethod of the present invention was able to almost completelydistinguish between O157:H7 and non-O157:H7 E. coli strains.

TABLE 6 Inclusivity Panel: 36 E. coli O157:H7 isolates Scorpion VersionPerosamine SIS1 Synthetase wbdr Sample Target 1 Target 2 Target 3 IDResult Result Result DD935 Positive Positive Positive DD1449 PositivePositive Positive DD1450 Positive Positive Positive DD1451 PositivePositive Positive DD1452 Positive Positive Positive DD1453 PositivePositive Positive DD1454 Positive Positive Positive DD1455 PositivePositive Positive DD1456 Positive Positive Positive DD1457 PositivePositive Positive DD1458 Positive Positive Positive DD1459 PositivePositive Positive DD1460 Positive Positive Positive DD1461 PositivePositive Positive DD1462 Positive Positive Positive DD1463 PositivePositive Positive DD1972 Positive Positive Positive DD1973 PositivePositive Positive DD1974 Positive Positive Positive DD1975 PositivePositive Positive DD1976 Positive Positive Positive DD1977 PositivePositive Positive DD1978 Positive Positive Positive DD1979 PositivePositive Positive DD1980 Positive Positive Positive DD1981 PositivePositive Positive DD1982 Positive Positive Positive DD1983 PositivePositive Positive DD1984 Positive Positive Positive DD1985 PositivePositive Positive DD1986 Positive Positive Positive DD1987 PositivePositive Positive DD1988 Positive Positive Positive DD1989 PositivePositive Positive DD1990 Positive Positive Positive DD1991 PositivePositive Positive

TABLE 7 Exclusivity Panel: 45 E. coli non-O157:H7 isolates ScorpionVersion Perosamine SIS1 Synthetase wbdr Sample Target 1 Target 2 Target3 ID Result Result Result DD1797 Negative — — DD1808 Negative NegativeNegative DD1809 Negative Negative Negative DD1821 Negative NegativeNegative DD1845 Negative Negative Negative DD1858 Negative NegativeNegative DD1859 Negative Negative Negative DD1869 Negative NegativeNegative DD1870 Negative Negative Negative DD1906 Negative NegativeNegative DD1915 Negative Negative Negative DD1927 Negative NegativeNegative DD1931 Negative Negative Negative DD2448 Negative NegativeNegative DD2450 Negative Negative Negative DD2453 Negative NegativeNegative DD2474 Negative Negative Negative DD2508 Negative NegativeNegative DD2511 Negative Negative Negative DD2514 Negative NegativeNegative DD2517 Negative Negative Negative DD2518 Negative NegativeNegative DD2522 Negative Negative Negative DD2523 Negative NegativeNegative DD3124 Negative Negative Negative DD3127 Negative NegativeNegative DD3130 Negative Negative Negative DD3132 Negative NegativeNegative DD3166 Negative Negative Negative DD3197 Negative NegativeNegative DD3199 Negative Negative Negative DD3204 Negative NegativeNegative DD3208 Negative Negative Negative DD3210 Negative NegativeNegative DD3785 Negative Negative Negative DD5884 Negative NegativeNegative DD5887 Negative Negative Negative DD5901 Negative NegativeNegative DD9705 Negative Negative Negative DD10910 Positive NegativeNegative DD12901 Negative Negative Negative DD5883 Negative NegativeNegative DD12800 Negative Negative Negative DD12804 Negative NegativeNegative DD12849 Negative Negative Negative DD12851 Negative NegativeNegative

Example 3 Determination of Inclusivity/Exclusivity of a Combination ofSIS1 and Wbdr Targets Via Scorpion Assay

Samples of organisms were analyzed to establish inclusivity andexclusivity of a combination of multiple Scorpion probes of the presentinvention. For inclusivity, independent, bona fide E. coli O157:H7isolates were used; for exclusivity non O157:H7 E. coli were used toensure that the assay would discriminate the target organism (O157:H7)from other E. coli. Inclusivity and exclusivity testing was performed onthe BAX® Q7 machine as described above.

Test Panel

The inclusivity panel was largely obtained from the Pennsylvania StateUniversity Department of Veterinary Science E. coli Reference Center andthe DuPont Qualicon culture collection. Isolates originated from a widerange of diagnostic and non-diagnostic samples. Inclusivity strains(n=61) included five strains of O157:H— non motile that were geneticallyO157:H7, one of the minor genetic Glade “cluster A”, and one of anO-rough phenotype that is genetically O157:H7. The exclusivity panel ofnon-E. coli, E. coli non-O157:H7, and E. coli O157 non-H7 strains (n=72)were chosen from the DuPont Qualicon culture collection. Most of theseisolates were originally obtained from naturally contaminated foodsamples or from animal sources, and all identifications were confirmedbio-chemically and/or serologically, as appropriate.

DNA Lysate Preparation

Cultures were struck for purity on BHI agar. For each strain, one colonywas inoculated into a tube containing BHI broth (exclusivity testing) ortest broth enrichment (inclusivity testing). Cultures were incubatedovernight at 35° C. to reach cell densities of approximately 10⁹bacterial cells per mL. For exclusivity testing, cultures were testedwith no dilution. For inclusivity testing, cultures were diluted to 10⁵cfu/mL, which is ˜1 log over the claimed sensitivity of the assay. 20 μlof the material to be tested was added to 200 μl of BAX® lysis reagent(DuPont Qualicon, Wilmington, Del.). The mixture was incubated at 37° C.for 20 minutes, then further incubated at 95° C. for 10 minutes, andfinally cooled to 5° C.

PCR Conditions

30 μl of the DNA lysate as prepared above was used to hydratelyophilized PCR reaction components to achieve a DNA lysate/PCR reactioncomponent mixture containing the primers and probes listed in Table 8.

TABLE 8 Quench- Per reaction 5′ Fluor er Primers wbdra2R 20 pMole (SEQID NO: 16) SIS1T75R 20 pMole (SEQ ID NO: 5) SV40-33-4312 20 pMole (SEQID NO: 27) SV40-29-4222 2.5 pMole  (SEQ ID NO: 28) Probes WBDR scorpion1 15 pMole Calflour BHQ1 (SEQ ID NO: 15) Orange 560 SIS1 scorpion 1  5pMole Quasar 670 BHQ2 (SEQ ID NO: 4) SV40 scorpion 1  5 pMole TAMRA BHQ2(SEQ ID NO: 29)SEQ ID NO:15 (WBDR scorpion 1 probe) is presented in the sequencelisting as the entire sequence without modifications. For this Example,the WBDR scorpion probe has, following nucleotide number 45 in SEQ IDNO:15, an internal BHQ1 quencher and an SP-18 blocker followed by theremaining 42 nucleotides. Similarly, SEQ ID NO:4 (SIS1 scorpion 1 probe)is presented in the sequence listing as the entire sequence withoutmodifications. For this Example, the SIS1 scorpion probe has, followingnucleotide number 46 in SEQ ID NO:4, an internal BHQ2 quencher and anSP-18 blocker followed by the remaining 40 nucleotides. Also, SEQ IDNO:29 (SV40 scorpion 1 probe) is presented in the sequence listing asthe entire sequence without modifications. For this Example, the SV40scorpion probe has, following nucleotide number 52 in SEQ ID NO:29, aninternal BHQ2 quencher and an SP-18 blocker followed by the remaining 26nucleotides.

The DNA lysate/PCR reaction component mixture was added to a PCRreaction mixture comprising typical PCR ingredients includingnucleotides, Taq polymerase and reaction buffer to perform PCR.

Amplification and testing was performed on the BAX® Q7 machine (DuPontQualicon, Wilmington, Del.). The thermal cycling conditions were: 2minutes at 94° C., followed by 40 cycles of 94° C. for 10 seconds and63° C. for 40 seconds, with the fluorescent signal captured during the63° C. step at each cycle.

Results

As shown in Tables 9 and 10, below, all isolates of E. coli O157:H7 gavepositive results, while all non-E. coli and E. coli which were notO157:H7 tested negative.

TABLE 9 Inclusivity panel: 61 E. coli O157:H7 isolates Strain AssayStrain Assay Number Strain Source Result Number Strain Source Result12836 E. coli PSU POS 12848 E. coli PSU POS O157:H7 ReferenceLab O157:H7Reference Lab 12830 E. coli PSU POS 12859 E. coli PSU POS O157:H7ReferenceLab O157:H7 Reference Lab 12832 E. coli PSU Reference POS 12860E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 12833 E. coli PSUReference POS 12861 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab12844 E. coli PSU Reference POS 12862 E. coli PSU POS O157:H7 LabO157:H7 Reference Lab 12845 E. coli PSU Reference POS 12863 E. coli PSUPOS O157:H7 Lab O157:H7 Reference Lab 12846 E. coli PSU Reference POS12874 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 12835 E. coliPSU Reference POS 12875 E. coli PSU POS O157:H7 Lab O157:H7 ReferenceLab 12834 E. coli PSU Reference POS 12876 E. coli PSU POS O157:H7 LabO157:H7 Reference Lab 12839 E. coli PSU Reference POS 12857 E. coli PSUPOS O157:H7 Lab O157:H7 Reference Lab 12840 E. coli PSU Reference POS12858 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 12841 E. coliPSU Reference POS 12869 E. coli PSU POS O157:H7 Lab O157:H7 ReferenceLab 12842 E. coli PSU Reference POS 12870 E. coli PSU POS O157:H7 LabO157:H7 Reference Lab 12843 E. coli PSU Reference POS 12871 E. coli PSUPOS O157:H7 Lab O157:H7 Reference Lab 12854 E. coli PSU Reference POS12873 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 12855 E. coliPSU Reference POS 12884 E. coli PSU POS O157:H7 Lab O157:H7 ReferenceLab 12856 E. coli PSU Reference POS 12885 E. coli PSU POS O157:H7 LabO157:H7 Reference Lab 12837 E. coli PSU Reference POS 12887 E. coli PSUPOS O157:H7 Lab O157:H7 Reference Lab 12849 E. coli PSU Reference POS12867 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 12850 E. coliPSU Reference POS 12868 E. coli PSU POS O157:H7 Lab O157:H7 ReferenceLab 12851 E. coli PSU Reference POS 12879 E. coli PSU POS O157:H7 LabO157:H7 Reference Lab 12852 E. coli PSU Reference POS 12880 E. coli PSUPOS O157:H7 Lab O157:H7 Reference Lab 12853 E. coli PSU Reference POS12881 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 12864 E. coliPSU Reference POS 12882 E. coli PSU POS O157:H7 Lab O157:H7 ReferenceLab 12865 E. coli PSU Reference POS 12883 E. coli PSU POS O157:H7 LabO157:H7 Reference Lab 12847 E. coli PSU Reference POS 12810 E. coli PSUPOS O157:H7 Lab O157:H7 Reference Lab 12813 E. coli PSU Reference POS12816 E. coli PSU POS O157:H7 Lab O157:H7 Reference Lab 2485 E. coliUnknown POS 8301 E. coli Unknown POS O157:HNM O157:HNM 5893 E. coliUnknown POS 8302 E. coli Unknown POS O157:HNM O157:HNM 5894 E. coliUnknown POS TD8136 E. coli Bovine POS O157:HNM O157:H7 Cluster A MA06 E.coli Peter Feng, POS O157:H7 FDA rough

TABLE 10 Exclusivity panel: 72 non-E. coli or E. coli non-O157:H7isolates Strain Assay Strain Assay Number Strain Source Result NumberStrain Source Result DD1081 Shigella boydii Unknown NEG DD2434 E. coliO1:H7 Unknown NEG DD11348 Enterobacter Unknown NEG DD2443 E. coliO157:H19 Unknown NEG DD1152 Listeria Pate' NEG DD2491 E. coli O2:H7Unknown NEG DD1261 Salmonella Duck NEG DD2520 E. coli O113:H7 UnknownNEG DD13249 Vibrio raw NEG DD2614 Edwardsiella Human feces NEG DD1716 E.coli O158:H23 Unknown NEG DD2901 Bacillus cereus Cream cake NEG DD1718E. coli O128:H2 Unknown NEG DD2992 Salmonella Unknown NEG DD1719 E. coliO28:HNM Unknown NEG DD3017 Salmonella Unknown NEG DD1720 E. coli O26:HNMUnknown NEG DD3019 Salmonella Unknnown NEG DD1721 E. coli O114:H32Unknown NEG DD3064 Morganella Environmental NEG DD1722 E. coli O127:HNMUnknown NEG DD3981 Enterococcus urine NEG DD1723 E. coli O119:H27Unknown NEG DD3982 Pseudomonas Blood culture NEG DD1724 E. coli O18:H14Unknown NEG DD3998 Streptococcus Bovine mastitis NEG DD1725 E. coliO125:H19 Unknown NEG DD4160 Staphylococcus Howler monkey NEG DD1777Salmonella Unknown NEG DD5588 Hafnia alvei Ground beef NEG DD1810 E.coli O28:H16 Unknown NEG DD577 Pseudomonas Human NEG DD1811 E. coliO127:H40 Unknown NEG DD5883 E. coli O55:H10 Unknown NEG DD1812 E. coliO127:H10 Unknown NEG DD610 Staphylococcus ham NEG DD1814 E. coli O6:H-Unknown NEG DD6121 Proteus mirabilis Gull, cloacal NEG DD1817 E. coliO29:H- Unknown NEG DD649 Listeria ivanovii sheep NEG DD1818 E. coliO136:H8 Unknown NEG DD6523 Klebsiella Ground beef NEG DD1819 E. coliO18:HNM Unknown NEG DD655 E. coli O101:K- Calf Intestine NEG DD1820 E.coli O86:H8 Unknown NEG DD661 Pseudomonas pre-filter tanks NEG DD1821 E.coli O55:H- Unknown NEG DD6719 Escherichia Sesame seeds NEG DD1822 E.coli O28:H8,4,3 Unknown NEG DD6832 Shigella sonnei Unknown NEG DD1824 E.coli O125:HNM Unknown NEG DD687 Lactobacillus vacuum NEG DD1825 E. coliO25:H8 Unknown NEG DD7005 Salmonella Unknown NEG DD1827 E. coli O20:HNMUnknown NEG DD7344 Lactobacillus Human NEG DD1831 E. coli O26:H11Unknown NEG DD846 Escherichia Cockroach NEG DD1833 E. coli O55:H9Unknown NEG DD847 Escherichia Human feces NEG DD1834 E. coli O29:H51Unknown NEG DD849 Escherichia Unknown NEG DD1835 E. coli O127:H- UnknownNEG DD850 Escherichia Human wound NEG DD1908 E. coli O25:H7 Unknown NEGDD922 Listeria innocua cured ham NEG DD2166 Salmonella Unknown NEGTD2631 Vibrio fluvialis Unknown NEG DD2274 Salmonella Unknown NEG TD3122Vibrio vulnificus Unknown NEG DD2341 Salmonella Unknown NEG TD3136Vibrio cholera Unknown NEG

Example 4 Comparative Sensitivity of Present Detection Methods Vs. USDAStandard Method in Beef Trim Sample

This study was performed to validate the ability of the present O157:H7detection test and methods to consistently detect 1-3 CFU of E. coliO157:H7 in a 375 gram sample of beef trim using a 1:5 sample to mediaratio.

Materials and Methods:

In each of three assay runs, twenty five 375 g beef trim samples werespiked at a target of 1.5 CFU per 375 g sample with E. coli O157:H7(strain DD1450 from DuPont collection). An unspiked media control wasrun as well on each run. Each run was performed on a different day.Spiking was performed using a single isolate of E. coli O157:H7 thatexhibits the phenotypes more commonly seen in E. coli O157:H7 (telluriteresistant, sorbitol non-fermenting, and sufficient expression of the Oantigen to render it routinely detectable by IMS prior to plating) inorder to simplify the confirmation process (plating on CT-SMAC) asdescribed by the United States Department of Agriculture's MicrobiologyLaboratory Guidebook (USDAMLG).

A single colony for the strain to be used was picked from a streakplate, used to inoculate 10 mL of Brain Heart Infusion (BHI) medium, andincubated at 37° C. for 24±2 h. This overnight growth was stored at 5°C. until use (24 to 48 hours) while serial dilutions were performed inpeptone water and the number of colony forming units determined byplating dilutions on BHI agar plates incubated at 37° C. for 24±2 h.Plates from dilutions with between 30 and 300 colonies were used for CFUper mL determination of the culture. Cultures were then serially dilutedto generate a target inoculum of 1.5 CFU/per inoculum volume with aninoculum volume between 10 and 1000 μL. Spike levels were confirmed attime of inoculation by triplicate plating of the sample inoculum volumefor each culture used on to BHI agar plates followed by incubation at37° C. for 18-24 hours and counting of colonies.

375 gram aliquots of trim cut as for n=60 surface sample analysis wereheld at 2-8° C. for 12-14 hours prior to inoculation. In addition,cultures were held at 2-8° C. for 24 hours prior to being used to spikebeef samples. Following spiking, bacteria were further cold stressed onthe meat for 18-24 hrs at 2-8° C. before start of enrichment. Spikedbeef samples were removed from refrigeration and held at roomtemperature for no more than 5 minutes prior to addition of 1.5 litersof BAX® MP media pre-warmed to 45±2° C. Following addition of media,samples were hand massaged to disperse trim fragments and the culturesmoved to an incubator held at 42±2° C. Samples were removed at timesindicated for testing.

At nine hours of incubation, samples were removed for testing. Lysatesof the enrichment broth were prepared and tested using both the newassay, as described below, and the standard United States Department ofAgriculture Microbiology Laboratory Guidebook (USDAMLG) PCR method (BAX®MP method).

Cells were grown overnight in BHI media. The cell lysate was prepared asfor the BAX® system E. coli multiplex (MP) test (as described in theBAX® user's guide, E.I. du Pont de Nemours & Co., Wilmington, Del.).Lysates were then used to hydrate tablets containing thermostablepolymerase, dNTPs, buffers, and salts and excipients suitable forcarrying out PCR. The reagents specific for detection of the targets inthis patent as well as the cycle conditions are described below inTables 11 (Taqman® assay) and 12 (Scorpion assay).

TABLE 11 Concen- Per 30 μl tration in Quench- reaction Reaction 5′ Fluorer Primers wbdRa1F 15 pMole 0.5 μM (SEQ ID NO: 17) wbdra2R 15 pMole 0.5μM (SEQ ID NO: 16) PerSt73F 12 pMole 0.4 μM (SEQ ID NO: 13) PerSt73R 12pMole 0.4 μM (SEQ ID NO: 12) SIS1T74F 12 pMole 0.4 μM (SEQ ID NO: 6)SIS1T75R 12 pMole 0.4 μM (SEQ ID NO: 5) Probes Perstalt166p  5 pMole0.17 μM  FAM BHQ1 (SEQ ID NO: 24) SIS1T65P 10 pMole 0.33 μM  Quasar BHQ2(SEQ ID NO: 25) 670 wbdRa1P 12.5 pMole  0.42 μM  CalFlour BHQ1 (SEQ IDNO: 26) Orange

For Table 8, the thermal cycling conditions were: 2 minutes at 94° C.,followed by 40 cycles of 94° C. for 15 seconds and 60° C. for 60seconds, with the fluorescent signal captured during the 60° C. step ateach cycle.

TABLE 12 Concen- Per 30 μl tration in Quench- reaction Reaction 5′ Fluorer Primers wbdra2R 20 pMole  0.5 μM (SEQ ID NO: 16) PerSt73R 20 pMole 0.4 μM (SEQ ID NO: 12) SIS1T75R 12 pMole  0.4 μM (SEQ ID NO: 5)Unimolecular Scorpion SV40 scorpion 1 10 pMole 0.33 μM TAMRA BHQ2 (SEQID NO: 29) SIS1 scorpion 1 10 pMole 0.33 μM Quasar BHQ2 (SEQ ID NO: 4)670 WBDR scorpion 1 15 pMole  0.5 μM CalFlour BHQ1 (SEQ ID NO: 15)Orange Bimolecular Scorpion PERSYNA probe 1 20 pMole 0.66 μM — BHQ1 (SEQID NO: 10 PERSYNA probe 1  5 pMole 0.17 μM 6FAM — (SEQ ID NO: 11

For Table 9, the thermal cycling conditions were: 2 minutes at 94° C.,followed by 40 cycles of 94° C. for 15 seconds and 60° C. for 40seconds, with the fluorescent signal captured during the 60° C. step ateach cycle.

At 24 hours of incubation all enrichments that had tested negative byeither the USDAMLG method or the method of the present invention wereremoved from the incubator and sampled for culture confirmation usingthe procedure as described in the USDA MLG.

Results:

Each sample is identified by run (A, B or C) followed by sample numberfor that run. For each run, numbers 1-25 represent samples that receivedan inoculum while number 26 is the media control blank. The targetedspike level for each run was 1.5 CFU/375 g beef sample. At this levelnot all samples would be expected to have actually received an inoculumcell due to poisson distribution. This is confirmed in Table 13, below,which gives the number of colonies on replicate plates each inoculatedwith 1× the inoculum used to spike the beef on that day and the numberof enrichments that were positive in the run.

TABLE 13 Number Plate 1 Plate 1 Plate 1 positive of (1X (1X (1X 25spiked Run inoculum) inoculum) inoculum) Average samples A 1 2 2 1.67 22B 2 0 2 1.33 20 C 1 3 2 2.0 22This data was used to generate a Most Probable Number (MPN) calculationin CFU/375 g sample for each run with 95% confidence intervals, as shownin Table 14 below.

TABLE 14 Number positive Calculated 95% Run of 25 MPN CI A 22 2.11.3-3.5 B 20 1.6 0.99-2.6  C 22 2.1 1.3-3.5The results of all of this testing is given in Table 15, below.

TABLE 15 USDAMLG Present standard Invention Culture Sample method Methodconfirmation A1 POSITIVE POSITIVE ND A2 NEGATIVE NEGATIVE ND A3 POSITIVEPOSITIVE ND A4 POSITIVE POSITIVE ND A5 POSITIVE POSITIVE ND A6 NEGATIVENEGATIVE NEGATIVE A7 POSITIVE POSITIVE ND A8 POSITIVE POSITIVE ND A9POSITIVE POSITIVE ND A10 POSITIVE POSITIVE ND A11 POSITIVE POSITIVE NDA12 POSITIVE POSITIVE ND A13 POSITIVE POSITIVE ND A14 POSITIVE POSITIVEND A15 NEGATIVE NEGATIVE NEGATIVE A16 POSITIVE POSITIVE ND A17 POSITIVEPOSITIVE ND A18 POSITIVE POSITIVE ND A19 POSITIVE POSITIVE ND A20POSITIVE POSITIVE ND A21 POSITIVE POSITIVE ND A22 POSITIVE POSITIVE NDA23 POSITIVE POSITIVE ND A24 POSITIVE POSITIVE ND A25 POSITIVE POSITIVEND A26 NEGATIVE NEGATIVE NEGATIVE B1 NEGATIVE NEGATIVE NEGATIVE B2NEGATIVE NEGATIVE NEGATIVE B3 POSITIVE POSITIVE * ND B4 POSITIVEPOSITIVE ND B5 POSITIVE POSITIVE ND B6 POSITIVE POSITIVE ND B7 POSITIVEPOSITIVE ND B8 POSITIVE POSITIVE ND B9 POSITIVE POSITIVE ND B10 POSITIVEPOSITIVE ND B11 POSITIVE POSITIVE ND B12 POSITIVE POSITIVE ND B13POSITIVE POSITIVE ND B14 POSITIVE POSITIVE ND B15 POSITIVE POSITIVE NDB16 POSITIVE POSITIVE ND B17 POSITIVE POSITIVE ND B18 POSITIVE POSITIVEND B19 POSITIVE POSITIVE ND B20 NEGATIVE NEGATIVE* NEGATIVE B21 POSITIVEPOSITIVE ND B22 NEGATIVE NEGATIVE NEGATIVE B23 POSITIVE POSITIVE ND B24POSITIVE POSITIVE ND B25 NEGATIVE NEGATIVE NEGATIVE B26 NEGATIVENEGATIVE NEGATIVE C1 POSITIVE Indeterminate* ND C2 POSITIVE POSITIVE NDC3 POSITIVE POSITIVE ND C4 POSITIVE POSITIVE ND C5 POSITIVE POSITIVE NDC6 POSITIVE POSITIVE ND C7 POSITIVE POSITIVE ND C8 POSITIVE POSITIVE NDC9 NEGATIVE NEGATIVE NEGATIVE C10 POSITIVE POSITIVE ND C11 POSITIVEPOSITIVE ND C12 POSITIVE POSITIVE ND C13 POSITIVE POSITIVE ND C14POSITIVE POSITIVE ND C15 POSITIVE POSITIVE ND C16 NEGATIVE NEGATIVENEGATIVE C17 POSITIVE POSITIVE ND C18 POSITIVE POSITIVE ND C19 POSITIVEPOSITIVE ND C20 POSITIVE POSITIVE ND C21 POSITIVE POSITIVE* ND C22POSITIVE POSITIVE ND C23 POSITIVE POSITIVE ND C24 NEGATIVE NEGATIVE NDC25 POSITIVE POSITIVE ND C26 NEGATIVE NEGATIVE NDFor the method of the present invention, a “Positive” sample was one inwhich all three markers used generated a positive result as called bythe instrument. “Indeterminate” results were those in which two of thethree markers generated a positive call by the instrument. One or notargets generating a positive call were Negative. * Indicates aninitially indeterminate call which was retested, with the result of theretest reported. One sample, C1, was indeterminate initially and uponretest. This was the only discrepancy with the USDAMLG method.Conclusion: The method of the present invention is able to reliablydetect even a single E. coli O157:H7 contaminant in a 375 g beef trimsample.

Example 5 Comparative Sensitivity of Present Detection Methods Vs. USDAStandard Method in Produce Samples

This study was performed to validate the ability of the present O157:H7detection test and methods to consistently detect 1-3 CFU of E. coliO157:H7 in a lettuce or spinach sample.

Sample Preparation

E. coli O157:H7 strains were grown overnight in BHI broth inoculatedwith a single colony. For the spinach sample trial, the strain employedwas DD1450, an E. coli O157:H7 strain within the DuPont Qualicon culturecollection that was isolated from a human clinical sample. For thelettuce trial, the strain employed was DD12835, an E. coli O157:H7strain obtained from the Pennsylvania State University Department ofVeterinary Science E. coli Reference Center.

Samples of sufficient quantity to perform all testing and Most ProbableNumber (MPN) analysis were inoculated with target diluted in sterile0.1% peptone water. Spike levels were set at levels likely to givefractional positive results (generally 1-3 cfu per analytical portion)and confirmed by plating from appropriate dilutions of the overnight BHIculture and MPN analysis on the day sample enrichment began.

Prior to inoculation, a sufficient portion was removed to perform allnecessary negative controls. Since the naturally occurring incidence ofE. coli O157:H7 is now so low in all matrices tested, no pre-screeningof matrix was conducted. The ten unspiked control samples were for eachreplicate (five test and five reference method samples) to demonstrateno naturally present target. None of these unspiked samples testedpositive.

Lettuce and spinach were purchased from local grocery stores. Theproduce was aseptically divided into portions for inoculation with thechallenge organism and an additional portion for a negative control.Forty 25 g test portions were surface inoculated at ˜1-3 cfu/25 g aswell as additional material to be tested by MPN and were recombined toform a master sample which was mixed well. Analytical units of 25 g wereremoved from this master sample and placed into stomacher bags. Afterinoculation, samples were stored at 4° C. for 2-3 days to adapt theorganism to the produce. An additional portion of inoculated sample wasused to determine the inoculation level using Most Probable Number (MPN)analysis (3×100 g, 3×10 g and 3×1 g). Ten 25 g portions of uninoculatedproduce were prepared as negative controls.

Comparative Method:

For the comparative test (FDA-BAM), produce samples (25 g) were diluted1:10 with EEB. Contents were mixed by stomaching briefly (10-30 sec) andallowed to incubate at 37±0.5° C. with shaking for 24±2 hr. Enrichedsamples were spread in 10-uL aliquot of undiluted enrichment onto TCSMACplate and in 0.1 mL aliquot of a 1:10 dilution onto another TCSMAC platefor isolation. All TCSMAC plates were incubated for 18-24 hr at 35-37°C., after which plates were examined for colonies with typicalcharacteristics of E. coli O157:H7. Suspect colonies (up to five perplate when present) were confirmed using the biochemical and serologicalmethods described in the FDA-BAM.

Test Method

For the method of the present invention, produce samples (25 g) werediluted with 225 mL pre-warmed BAX® System E. coli O157:H7MP broth andincubated for 24 hr at 42° C.±2° C., sampling at 8, 10, and 24 hourswith BAX® System assay. All samples, without respect to presumptiveresult, were subjected to culture confirmation as in the referencemethod.

Cultures were struck for purity on BHI agar. For each strain, one colonywas inoculated into a tube containing BHI broth (exclusivity testing) ortest broth enrichment (inclusivity testing). Cultures were incubatedovernight at 35° C. to reach cell densities of approximately 10⁹bacterial cells per mL. For exclusivity testing, cultures were testedwith no dilution. For inclusivity testing, cultures were diluted to 10⁵cfu/mL, which is ˜1 log over the claimed sensitivity of the assay. 20 μlof the material to be tested was added to 200 μl of BAX® lysis reagent(DuPont Qualicon, Wilmington, Del.). The mixture was incubated at 37° C.for 20 minutes, then further incubated at 95° C. for 10 minutes, andfinally cooled to 5° C.

30 μl of the DNA lysate as prepared above was used to hydratelyophilized PCR reaction components to achieve a DNA lysate/PCR reactioncomponent mixture containing the primers and probes listed in Table 16.

TABLE 16 Quench- Per reaction 5′ Fluor er Primers wbdra2R 20 pMole (SEQID NO: 16) SIS1T75R 20 pMole (SEQ ID NO: 5) SV40-33-4312 20 pMole (SEQID NO: 27) SV40-29-4222 2.5 pMole  (SEQ ID NO: 28) Probes WBDR scorpion1 15 pMole Calflour BHQ1 (SEQ ID NO: 15) Orange 560 SIS1 scorpion 1  5pMole Quasar 670 BHQ2 (SEQ ID NO: 4) SV40 scorpion 1  5 pMole TAMRA BHQ2(SEQ ID NO: 29)SEQ ID NO:15 (WBDR scorpion 1 probe) is presented in the sequencelisting as the entire sequence without modifications. For this Example,the WBDR scorpion probe has, following nucleotide number 45 in SEQ IDNO:15, an internal BHQ1 quencher and an SP-18 blocker followed by theremaining 42 nucleotides. Similarly, SEQ ID NO:4 (SIS1 scorpion 1 probe)is presented in the sequence listing as the entire sequence withoutmodifications. For this Example, the SIS1 scorpion probe has, followingnucleotide number 46 in SEQ ID NO:4, an internal BHQ2 quencher and anSP-18 blocker followed by the remaining 40 nucleotides. Also, SEQ IDNO:29 (SV40 scorpion 1 probe) is presented in the sequence listing asthe entire sequence without modifications. For this Example, the SV40scorpion probe has, following nucleotide number 52 in SEQ ID NO:29, aninternal BHQ2 quencher and an SP-18 blocker followed by the remaining 26nucleotides.

The DNA lysate/PCR reaction component mixture was added to a PCRreaction mixture comprising typical PCR ingredients includingnucleotides, Taq polymerase and reaction buffer to perform PCR.

Amplification and testing was performed on the BAX® Q7 machine (DuPontQualicon, Wilmington, Del.). The thermal cycling conditions were: 2minutes at 94° C., followed by 40 cycles of 94° C. for 10 seconds and63° C. for 40 seconds, with the fluorescent signal captured during the63° C. step at each cycle.

At 24 hours of incubation, all enrichments that had tested negative byeither the USDA MLG method or the method of the present invention wereremoved from the incubator and sampled for culture confirmation usingthe procedure as described in the USDA MLG.

Data Analysis:

Data analysis was performed according to the AOAC guidelines formicrobiological method validation. Most probable number (MPN) of colonyforming units per test portion was performed on the day of testing usingthe reference method. MPN values were calculated using the tables foundin the FDA-BAM. Spike level was determined by performing a standardplate count on the incident cultures which were diluted for spiking eachmatrix on the day of introduction to the master sample. Sensitivity rateis calculated as 100 times the number of true presumptive positiveresults divided by total true positive results confirmed from enrichmentof spiked samples. False negative rate was calculated as 100 minussensitivity rate. Specificity rate was calculated as 100 times thenumber of assay-negative results divided by total number of truenegative results, including unspiked samples. False positive rate wascalculated as 100 minus specificity rate. A Chi square test forsignificant difference was performed using the McNemar formula:(|a−b|−1)²/(a+b), where a=results that were positive by BAX and negativeby reference method, and b=results that were negative by BAX andpositive by reference method used for paired samples, Mantel Haenszelfor unpaired samples. A Chi square value of less than 3.84 indicates nosignificant difference, at the 95% confidence level, between the twomethods, while a Chi square value of greater than 3.84 indicates asignificant difference between the test and reference methods.

RESULTS AND CONCLUSION

As shown in Tables 17 and 18, below, using produce enrichments (lettuceand spinach) as samples, the test method demonstrated equivalent orsuperior performance relative to the reference method.

TABLE 17 Results of 25 g Lettuce Spiked with Strain DD12835 MPNEnrichment Presump Presump. False Per Spike Method Total Pos/Sensitivity False Pos/ Specificity Pos Chi- Method 25 g Level (Media)spiked Confirmed % Neg % Unspiked % % square  8 hr BAX 1.1 1.0 BAX MP 2015/16  94 6 0/5 100 0 6.3 10 hr BAX 1.1 1.0 BAX MP 20 15/16  94 6 0/5100 0 6.3 24 hr BAX 1.1 1.0 BAX MP 20 16/16 100 0 0/5 100 0 8.1Reference 1.1 1.0 EEB 20 7 NA NA 0/5 NA NA (24 hr)

TABLE 18 Results of 25 g Spinach Spiked with Strain DD1450 MPNEnrichment Presump Presump. Per Spike Method Total Pos/ SensitivityFalse Pos/ Specificity False Chi- Method 25 g Level (Media) spikedConfirmed % Neg % Unspiked % Pos % square  8 hr BAX 0.23 1.0 BAX MP 2012/13  92 8 0/5 100 0 3.6 10 hr BAX 0.23 1.0 BAX MP 20 13/13 100 0 0/5100 0 4.8 24 hr BAX 0.23 1.0 BAX MP 20 13/13 100 0 0/5 100 0 4.8Reference 0.23 1.0 EEB 20 6 NA NA 0/5 NA NA (24 hr)

1-10. (canceled)
 11. An isolated polynucleotide comprising SEQ ID NO:4,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, or SEQ IDNO:26.
 12. The isolated polynucleotide of claim 11, wherein saidpolynucleotide comprises a primer region and a probe region, whereinsaid polynucleotide further comprises an 18-carbon non-amplifiablelinker joining said primer region to said probe region, and wherein saidpolynucleotide further comprises a detectable label. 13-21. (canceled)